Disturbances of Growth Control- · bind Pro-rich regions ... 2012-2020 GIST patient w abdominal mets before Glivec treatment 48 hr after Glivec 8 days after Glivec Positron Emission

Disturbances of Growth ControlDisturbances of Growth Control--& how to target them for therapy& how to target them for therapy

June 9, 2006June 9, 2006

Nancy HynesNancy HynesFriedrich Friedrich MiescherMiescher InstituteInstitute

SS07 #13704SS07 #13704

In the past 25 yrs cancer research has generated a largebody of knowledge on molecular mechanisms controlling allaspects of normal cellular physiology/signaling pathways.

These studies have also told us a lot about the molecularmechanisms underlying cancer development.

Goal – to design rational/molecularly targeted therapeutics for cancer treatment.

The signaling circuitry in a normal cell is integrated,The signaling circuitry in a normal cell is integrated,finely regulated and in tune with the external milieufinely regulated and in tune with the external milieu

Hanahan & Weinberg, Cell, 2000

Hanahan & Weinberg (2000) Cell

http://www.cell.com/content/vol100/issue1/images/large/CELL.100_1_57.1112.f2.jpeg

Normal cells are strictly dependent upon external cuesNormal cells are strictly dependent upon external cues

DifferentiateDifferentiate

DieDie

Divide/restDivide/rest



The signaling circuitry in a cancer cell has undergoneThe signaling circuitry in a cancer cell has undergonemany changes many changes --

Red proteins:Mutated, lost oroverexpressed in cancer cells.

The circuitry in a tumor cell is The circuitry in a tumor cell is integrated, but no longer responsive to integrated, but no longer responsive to normal external cuesnormal external cues



However, However, underlying principlesunderlying principles have begun to emerge and thesehave begun to emerge and theseprovide an important framework for understanding cancer andprovide an important framework for understanding cancer andfor designing therapies. for designing therapies.

In the past 25 yrs cancer research has generated a largeIn the past 25 yrs cancer research has generated a largebody of knowledge on molecular mechanisms controlling allbody of knowledge on molecular mechanisms controlling allaspects of normal cellular physiology. These studies have alsoaspects of normal cellular physiology. These studies have alsotold us a lot about the molecular mechanisms underlying told us a lot about the molecular mechanisms underlying cancer development. cancer development.

The apparent complexity makes the problem seem almostThe apparent complexity makes the problem seem almostunmanagableunmanagable! !


Acquired Capabilities of Cancer CellsAcquired Capabilities of Cancer Cells

Coussens & Werb (2002) Nature 420:860

Cancer cells are influenced by their environment Cancer cells are influenced by their environment

Multiple cell types in the tumor environment Multiple cell types in the tumor environment

FibroblastsFibroblasts –– extracellularextracellular matrixmatrixMacrophagesMacrophages –– ligandligand production, e.g. VEGFproduction, e.g. VEGFEndothelial cellsEndothelial cells –– provide oxygen & growth factorsprovide oxygen & growth factors

--attract tumor cells to the blood & lymphattract tumor cells to the blood & lymphvesselsvessels

Traits for malignancy promoted by macrophages

Condeelis & Pollard (2006) Cell

StressStressDNA damageDNA damageoncogenesoncogenes

Gain of function & loss of function mutations Gain of function & loss of function mutations contribute to the cancer phenotypecontribute to the cancer phenotype

Gain of function = Gain of function = oncogenesoncogenes

Loss of function = Loss of function = tumor suppressorstumor suppressors

Growth control pathways & cancerGrowth control pathways & cancer

The The RasRas/ERK & PI3K pathways are altered in most cancers. /ERK & PI3K pathways are altered in most cancers.

Shaw & Cantley (2006) Nature 441:424

Tumor Suppressors/Loss of Function MutationsTumor Suppressors/Loss of Function Mutations

Some tumor suppressors are lost in many cancer types:Some tumor suppressors are lost in many cancer types:PTEN, p53, PTEN, p53, RbRb

Loss of some tumor suppressors leads to cancer in specific organLoss of some tumor suppressors leads to cancer in specific organs:s:BRCA, APC, hMSH2, hMLH1, LKB1, VHLBRCA, APC, hMSH2, hMLH1, LKB1, VHL

Control functions/pathways important in many cell typesControl functions/pathways important in many cell types

Control cell specific functions Control cell specific functions

BRCA = breast cancer associated – breast/ovary cancerAPC = adenomatous polyposis coli – colon cancerMSH2 & MLH1 = DNA repair proteins – colon cancerLKB1 = ser/thr protein kinase 11 – GI tract polyps VHL = Von Hipple-Lindau – kidney cancer

Tumor SuppressorsTumor Suppressors

Tumor suppressor genes can be inherited:Tumor suppressor genes can be inherited:

1 mutant allele predisposes to specific tumor types1 mutant allele predisposes to specific tumor types22ndnd allele is lost during tumor developmentallele is lost during tumor development

Loss of tumor suppressor genes occurs in sporadic tumors:Loss of tumor suppressor genes occurs in sporadic tumors:

both copies are lost/mutated during cancer developmentboth copies are lost/mutated during cancer developmentusuallyusually inherited genes are also involved in sporadic cancerinherited genes are also involved in sporadic cancer

Loss of tumor suppressor genes relieve pathwaysLoss of tumor suppressor genes relieve pathwaysthat blocks cancer development.that blocks cancer development.

Retroviruses & CancerRetroviruses & Cancer

Proviral DNAProviral DNA: : -- encodes an oncogene encodes an oncogene -- activates an activates an oncogeneoncogene upon integration upon integration

RNARNA--containing virusescontaining virusesRSV MMTVMC29, SSV, Ha-MSVAEVEtc.

Rous Sarcoma VirusRous Sarcoma Virus

RSV is a replication competent virus encoding the v-src oncogene.

1911 - Isolated from a chicken fibrosarcoma by Peyton Rous.

1966 - Rous received the Nobel Prize for his discovery.

1977 - M.Bishop & H.Varmus - v-src related to c-src –our DNA encodes oncogenes! Nobel Prize in 1989.

c-src was “captured” by a retrovirus

SrcSrc characteristics characteristics

First member of the c-src family of cytoplasmic proteintyrosine kinases.

Domains promoting protein-protein interaction.

Src Homology domains = SH

SH2

SH3 Bind PBind P--TyrTyr Bind ProBind Pro--rich domainsrich domains

SH1 – kinasedomain

PP--Tyr 527 binds the SH2 domain of Tyr 527 binds the SH2 domain of SrcSrcmaintaining the maintaining the kinasekinase in an inactive state. in an inactive state.

AutoinhibitionAutoinhibition of of kinasekinase activityactivity

SH2 domains SH2 domains bind Pbind P--Tyr Tyr

SH3 domains SH3 domains bind Probind Pro--rich regions rich regions

CC--terminal terminal srcsrc kinasekinase = = CskCskPhosphorylatesPhosphorylates TyrTyr--527527

cc--SrcSrc is activated by various mechanismsis activated by various mechanisms

cc--SrcSrc activity is higher activity is higher insomeinsome human cancers (breast & colon). human cancers (breast & colon).

(STAT lecture (STAT lecture –– B. B. GronerGroner June 30June 30thth) )

Mouse Mammary Tumor Virus Mouse Mammary Tumor Virus -- MMTVMMTV

Transmitted in the milk of certain mouse strains (C3H).

Female mice have a high incidence of mammary tumors.

MMTV acts via insertional mutagenesis.

The first identified MMTV integration site was upstream of the int-1 gene (Wnt-1).

IntInt--1 = Wnt1 = Wnt--1, 1, an activator of the an activator of the ββ--catenin/Tcf pathwaycatenin/Tcf pathway

This pathway is activated in human colorectal tumorsmainly via mutation of APC. (Discussed in Summer 2005)

APC

HH--rasras -- the first the first oncogeneoncogenediscovered in humandiscovered in humantumor DNAtumor DNA

BarbacidBarbacid, Weinberg,, Weinberg,WiglerWigler grpsgrps ~1980~1980

T24 bladder T24 bladder cancer cell linecancer cell line

11stst slideslide

RasRas is mutated in manyis mutated in manyhuman tumors. human tumors.

KK--rasras or Nor N--rasras

2nd Slide2nd Slide

HH--rasras -- the first the first oncogeneoncogenediscovered in humandiscovered in humantumor DNAtumor DNA

Harvey Harvey MurineMurine Sarcoma Virus Sarcoma Virus –– HaHa--MSVMSV

Encodes the HEncodes the H--rasras oncogeneoncogene

RasRas encoding retroviruses have been isolated from mice, encoding retroviruses have been isolated from mice, rats and cats.rats and cats.

HH--rasras isolated from bladder tumor DNA had the same isolated from bladder tumor DNA had the same mutation found in viral Hmutation found in viral H--RasRas. .

RasRas proteins are proteins are ““G proteinsG proteins””that cycle between an inactive that cycle between an inactive GDPGDP--bound form and an activebound form and an activeGTPGTP--bound form.bound form.

OncogenicOncogenic RasRas proteins (viral or human) have an activating mutationproteins (viral or human) have an activating mutationthat prevents GAP from hydrolyzing GTP. that prevents GAP from hydrolyzing GTP.

Active Active RasRas is always is always ““onon”” &&signaling to its downstreamsignaling to its downstreameffectoreffector proteins.proteins.

Many classes of Many classes of RTKsRTKs are aberrantly activated in are aberrantly activated in human cancerhuman cancer

ErbB1ErbB1EGFREGFR

ErbB2ErbB2NeuNeuHER2HER2

ErbB3ErbB3HER3HER3

ErbB4ErbB4HER4HER4

Epidermal growth factor receptor & human cancer Epidermal growth factor receptor & human cancer

Avian Avian erythroblastosiserythroblastosis virus (AEV) virus (AEV)

Avian retrovirus which encodes two Avian retrovirus which encodes two oncogenesoncogenes::ErbAErbA and and ErbBErbB..

ErbAErbA is a mutated is a mutated ligandligand--independent version ofindependent version ofthe thyroid hormone receptor (T3).the thyroid hormone receptor (T3).

ErbBErbB is a mutated is a mutated ligandligand--independent version ofindependent version ofthe EGF receptor.the EGF receptor.

Concept of cooperating Concept of cooperating oncogenesoncogenes –– few if any few if any examples of a tumor arising from a single alteration.examples of a tumor arising from a single alteration.

Mechanisms leading to RTK activation in cancer:Mechanisms leading to RTK activation in cancer:using the ERBB receptors as an exampleusing the ERBB receptors as an example

Gene amplification & receptor Gene amplification & receptor overexpressionoverexpression

Mutations Mutations ––KinaseKinase Domain or ECDDomain or ECD

NRGARTGFα

Stromal cells

AutocrineAutocrine or or paracrineparacrine activation due to activation due to ligandligand availabilityavailability

Paul Ehrlich (1854Paul Ehrlich (1854--1915)1915)

1908 Nobel Prize1908 Nobel Prize

Targeting Cancer with Targeting Cancer with ““Magic BulletsMagic Bullets””

Ehrlich’s work on aniline dyes led to the concept that chemical substances might have special affinities for pathogenic organisms & cancer cells.

These dyes, referred to as “Magic Bullets”would go straight to organisms or cells &kill them.

AlkylatingAlkylating drugs were the 1drugs were the 1stst antianti--cancer agents usedcancer agents usedin the clinic & ~1940s in the clinic & ~1940s –– some success in lymphoma treatment.some success in lymphoma treatment.

Other types of chemotherapeutic drugs were developedOther types of chemotherapeutic drugs were developedin the 1950in the 1950--1960s 1960s –– drugs designed to block drugs designed to block purinepurine & & pyrimidinepyrimidine metabolism.metabolism.

Targeted antiTargeted anti--cancer drugs that block a cellular process, cancer drugs that block a cellular process, for example, DNA synthesis. for example, DNA synthesis.

Targeting Cancer with Targeting Cancer with ““Magic BulletsMagic Bullets””

Today antiToday anti--cancer drugs target specific proteins/pathwayscancer drugs target specific proteins/pathwaysknown to be altered in cancer cells. known to be altered in cancer cells.

(More history in – H.Varmus 2006 Science 312: 1162)

Inhibitors targeting different classes of Inhibitors targeting different classes of RTKsRTKs are are in the clinic or in advanced stages of developmentin the clinic or in advanced stages of development

EGFREGFR;;IressaIressaTarcevaTarceva

PDGFR/KitPDGFR/KitGleevecGleevec

FLT3FLT3::PKC412PKC412

VEGFRVEGFR::AvastinAvastinPTK787PTK787ZD6474ZD6474SU6668SU6668

FGFRFGFR::PD173074PD173074

Ret etc.Ret etc.

Sebolt-Leopold & English (2006) Nature 441: 457

KinaseKinase targeted antitargeted anti--cancer therapeuticscancer therapeutics

FDA Approved FDA Approved

KinaseKinase targeted antitargeted anti--cancer therapeutics in cancer therapeutics in development development


mTORmTORCCICCI--779; RAD001779; RAD001PhosphoinositidePhosphoinositide kinasekinase--related related kinasekinase (PIKK) (PIKK)

PDGFR Family Members & Cancer PDGFR Family Members & Cancer

Gleevec/imatinibGleevec/imatinib

Hernandez et al (2004) Trends in Call Biology 14:36

BCRBCR--AblAbl is constitutively active in CML; many experiments is constitutively active in CML; many experiments suggested that blocking suggested that blocking AblAbl kinasekinase activity would kill activity would kill leukemicleukemic cells. cells.

CML patients show a balanced translocation CML patients show a balanced translocation ––Philadelphia Chromosome described by Nowell & Hungerfordin the 1960s - encodes the BCR-Abl oncoprotein

Targeting BCRTargeting BCR--ABL in Chronic ABL in Chronic MyelogenousMyelogenous Leukemia Leukemia

The TKI The TKI imatinib/Glivecimatinib/Glivec was found to reverse the was found to reverse the cllinicalcllinicalsymptoms of CML symptoms of CML –– the Philadelphia Chromosome+ cells were lost. the Philadelphia Chromosome+ cells were lost.

First cancer in which a TKI was found effective. First cancer in which a TKI was found effective.

PDGFR Family Members & Cancer PDGFR Family Members & Cancer

PDGFR PDGFR –– ligandligand activated (lung tumors)activated (lung tumors)Kit Kit –– mutationsmutationsFlt3 Flt3 –– mutations (mutations (leukemiasleukemias) (PKC412) ) (PKC412)

Gleevec/imatinibGleevec/imatinib also targets the PDGF & Kit receptoralso targets the PDGF & Kit receptor

Kit Receptor & CancerKit Receptor & Cancer

Gastrointestinal Gastrointestinal stromalstromal tumors (GIST) are rare, tumors (GIST) are rare, however, they are the most frequent however, they are the most frequent mesenchymalmesenchymaltumors of the GI tract. tumors of the GI tract.

The majority of The majority of GISTsGISTs have activating mutations in thehave activating mutations in thecc--Kit receptor. Kit receptor. (Rubin et al 2001 Cancer Res., 61: 8118)(Rubin et al 2001 Cancer Res., 61: 8118)

GleevecGleevec blocks cblocks c--Kit Kit kinasekinase activity. activity. ((BuchdungerBuchdunger et al., 2000 J. et al., 2000 J. PharmacolPharmacol. Exp. . Exp. TherTher., 295: 139., 295: 139--145)145)

Complete Response to Complete Response to GleevecGleevec in a GIST patientin a GIST patient

StroobantsStroobants et al., 2003 et al., 2003 EurEur. J.Cancer 39: 2012. J.Cancer 39: 2012--20202020

GIST patient w abdominal metsbefore Glivec treatment

48 hr after Glivec 8 days after Glivec

Positron Emission Tomography (PET) with Positron Emission Tomography (PET) with 1818F F fluorodeoxyglucosefluorodeoxyglucose..

FGFR & CancerFGFR & Cancer

Gene amplification and/or Gene amplification and/or overexpressionoverexpression ofofreceptors occur in a high % of breast cancers.receptors occur in a high % of breast cancers.

20% FGFR20% FGFR--1155--10% FGFR10% FGFR--22

30% FGFR30% FGFR--44

Other cancers with high expression of Other cancers with high expression of FGFRsFGFRs::prostate cancerprostate cancergastric cancergastric cancercolorectal cancercolorectal cancerovarian cancerovarian cancer

FGFR

PPPPP P

PD

P

signaling

PD173074 is a selective FGFR PD173074 is a selective FGFR kinasekinase inhibitorinhibitor

PTyr

FGFR-4

SUM 52

PTyr

FGFR-2

SUM 52MDA-MB-453

- + - + - +- +MDA-MB-415

IP FGFR-4 IP FGFR-2

PD

PD173074 blocks FGFR phosphorylation in breast cancer cellsPD173074 blocks FGFR phosphorylation in breast cancer cells

KoziczakKoziczak et al., 2004 et al., 2004 OncogeneOncogene 23: 350123: 3501

MDA-MB-453

MCF10A

MDA-MB-415

SUM 52

day 0 day 4

day 0 day 4

day 0 day 4 day 8

day 0 day 4 day 8

day 8

day 8

DMSOPD173074

PD173074 blocks proliferation of cancer cells PD173074 blocks proliferation of cancer cells with active FGFR with active FGFR

MCF10A MCF10A –– normalnormalbreast cellsbreast cells

FGFRsFGFRs appear to be good therapeutic targets in breast cancer.appear to be good therapeutic targets in breast cancer.

KoziczakKoziczak et al., 2004 et al., 2004 OncogeneOncogene 23: 350123: 3501

proliferation, survival, migration & metastasisproliferation, survival, migration & metastasis

PPPPP

PPPPP

PPPPP

PPPPP

MAPK

AKT

STAT

SRC

mTOR

PPPPPPPPPP

PPPPP

PPPPP

PPPPP

PPPPPPPPPP

Nuclear CompartmentNuclear Compartment

Cytoplasmic DomainCytoplasmic DomainPPPPP

PPPPPPPPPPPPPPP

PPPPP PPPPPPI3K

ExtracellularExtracellular DomainDomain

Plasma MembranePlasma Membrane

ERBB Receptors

X

Z

ERBB targeted antibodies:HerceptinCetuximab

TKI

TKI

ERBB targeted tyrosine kinaseinhibitors (TKIs):GefitinibErlotinib

ERBB ERBB targetedtargeted inhibitorsinhibitors

Important conceptImportant concept

Despite the fact that individual tumors displayDespite the fact that individual tumors displaydifferent mutations, the pathways causing transformation different mutations, the pathways causing transformation of a normal cell into a cancer cell are often similar.of a normal cell into a cancer cell are often similar.

PI3K & MAPK pathwayPI3K & MAPK pathwayare active in most humanare active in most humantumors.tumors.



The PI3K/AKT pathway is altered in most cancers. The PI3K/AKT pathway is altered in most cancers.


PI3K/PKB pathwayPI3K/PKB pathway

PI3K catalyzes the production of PI3K catalyzes the production of phosphatidylinositolphosphatidylinositol 3,4,53,4,5--triphosphatetriphosphate(PIP3) at the cell membrane.(PIP3) at the cell membrane.

PKB/Akt has two upstream PKB/Akt has two upstream kinaseskinasesrequired for its activation.required for its activation.

Ser308Ser308

Ser473Ser473

PI3K/PKB pathwayPI3K/PKB pathway

PIP3 recruits Akt to the membrane via its PH domainPIP3 recruits Akt to the membrane via its PH domain

PKB/Akt has many effectorsPKB/Akt has many effectors


BadBad--PPBcl2 is free Bcl2 is free to promote to promote survivalsurvival

FOXOFOXO--PPp27 p27 cyclinE/Cdk2cyclinE/Cdk2

GSKGSK--33--PPCyclin D Cyclin D proliferationproliferation

AKT8 retrovirus isolated from AKT8 retrovirus isolated from thymomasthymomas in mice. in mice.

Retroviruses & the Retroviruses & the phosphoinositidephosphoinositide 33’’--kinase pathwaykinase pathway

The The oncogeneoncogene vv--Akt Akt -- encoded as a gagencoded as a gag--fusion proteinfusion proteinwith serinewith serine--threoninethreonine kinasekinase activity.activity.BellacosaBellacosa et al (1991) Science 254: 274et al (1991) Science 254: 274--277.277.

Stall,S (1987) PNAS 84: 5034Stall,S (1987) PNAS 84: 5034--50375037

The avian sarcoma virus 16 (ASV16) encodes an activeThe avian sarcoma virus 16 (ASV16) encodes an activeversion of the catalytic subversion of the catalytic sub--unit of PI 3unit of PI 3--kinase.kinase.

Chang et al (1997) Science 276: 1848.Chang et al (1997) Science 276: 1848.

The PI3K/PKB pathwayThe PI3K/PKB pathway

Somatic mutations in Somatic mutations in PIK3CAPIK3CA, which encodes the p110, which encodes the p110αα catalyticcatalyticsubsub--unit are found in many human tumors.unit are found in many human tumors.

Samuels et al., April 23, 2004, Science 304, 554.Samuels et al., April 23, 2004, Science 304, 554.

These mutations are predicted to lead to constitutiveThese mutations are predicted to lead to constitutiveactivity of the p110activity of the p110αα catalytic subcatalytic sub--unit.unit.







The PI3K pathway & PTENThe PI3K pathway & PTEN

PTEN was identified as a tumor suppressor gene frequently PTEN was identified as a tumor suppressor gene frequently deleted in advanced tumors.deleted in advanced tumors.Li et al. 1997 Science 275: 1943; Li et al. 1997 Science 275: 1943; SteckSteck et al 1997 Nat Genet 15: 356et al 1997 Nat Genet 15: 356

~ 50% of advanced human tumors carry inactivating mutations ~ 50% of advanced human tumors carry inactivating mutations in PTEN.in PTEN.

PTEN mutations are rare in breast cancer; LOH & promoterPTEN mutations are rare in breast cancer; LOH & promotermethylationmethylation leading to low expression of PTEN are common.leading to low expression of PTEN are common.

SHIP = SHIP = SHSH2 containing 2 containing iinositolnositol 55’’ pphosphatasehosphatase

PTEN = PTEN = pphosphatasehosphatase & & tentensinsin


PTEN is a lipid PTEN is a lipid phosphatasephosphatase that antagonizes the action of PI3K by that antagonizes the action of PI3K by removing the 3removing the 3--phosphate from PtdIns(3,4,5)Pphosphate from PtdIns(3,4,5)P3.3.

PIP3PIP3


PTEN PTEN downregulatesdownregulates a a kinasekinase cascade with important cellularcascade with important cellularmediators. mediators.

VivancoVivanco & Sawyers 2002 & Sawyers 2002 volvol 2, 4892, 489

PIP3PIP3 PIP2PIP2

The PI3K/PKB pathwayThe PI3K/PKB pathway

Active in most human tumorsActive in most human tumors

due to loss of negative regulatorsdue to loss of negative regulators

inactivating mutations in PTENinactivating mutations in PTEN

due to activating mutationsdue to activating mutations

catalytic subcatalytic sub--unit p110unit p110

LST8

LST8

RaptorRaptor

Alessi et al 2006 Ann Rev Biochem 75:137

mTORmTOR is controlled by growth factors & energy is controlled by growth factors & energy

Energy/stress/hypoxiaEnergy/stress/hypoxia

mTORmTOR = = mammalianmammalian targettarget of of rapamycinrapamycin

mTORmTOR is a member of the is a member of the phosphoinositidephosphoinositide--kinasekinase--related related kinasekinase family (PIKK). family (PIKK).

mTORmTOR acts as a central sensor for nutrientsacts as a central sensor for nutrients& energy.& energy.

LST8

LST8

RaptorRaptor


mTORmTOR is controlled by growth factors & energy is controlled by growth factors & energy

PI3K activating mutations (p110PI3K activating mutations (p110αα))found in ~25% primary breast found in ~25% primary breast tumors. tumors.

Loss of PTEN (LOH) orLoss of PTEN (LOH) orpromoter promoter methylationmethylation--~30% breast tumors~30% breast tumors

mTOR

S65’

3’

40S

60S

40S

60S

S65’

3’P

4E-BP1eIF-4E

P P

eIF-4E4E-BP1P P P P

+

S6K1P P

ControlControl translationtranslation of of growthgrowth--relatedrelated mRNAsmRNAs

mTORmTOR controls translation of growthcontrols translation of growth--related mRNAsrelated mRNAs

Ribosomal proteins, cell cycle regulators (cyclin D) etc Ribosomal proteins, cell cycle regulators (cyclin D) etc ––proteins needed for tumor cell growth & proliferationproteins needed for tumor cell growth & proliferation

RapamycinRapamycin

FKBP12FKBP12RapamycinRapamycin blocks blocks mTORmTOR/Raptor /Raptor complex activitycomplex activity

40SS6

5’3’

60S

5’3’

40SS6

60S

5’3’

40SS6

Monosome

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

Disome

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

Trisome

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6

60S

40SS6 Polysome

60S

40SS6

40SS6

40SS6

60S

60S

60S

RibosomalSubunits

40SS6

5’3’

RapamycinRapamycin activity can be activity can be examined on examined on polysomepolysome profilesprofiles

RNA extraction

Northern Blot

QuantificationPolysome profile

Polysomefractions

80S

60S

40S

0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11

polysomal fractionsnon-polysomal

Relative

amou

nt

of c

yclin

D1

mRN

A (%)

polysomal fractionsnon-polysomal

Examine Examine polysomalpolysomal distribution of mRNA of interestdistribution of mRNA of interest

Cyclin D1 mRNA translation is sensitive to Cyclin D1 mRNA translation is sensitive to rapamycinrapamycin

KoziczakKoziczak & Hynes 2004 JBC 279:50004& Hynes 2004 JBC 279:50004

RapamycinRapamycin treated tumor cells have very low levels of cyclin D1 & treated tumor cells have very low levels of cyclin D1 & block in the G1 phase of the cell cycle.block in the G1 phase of the cell cycle.






BRCA = breast cancer associated – breast/ovary cancerAPC = adenomatous polyposis coli – colon cancerMSH2 & MLH1 = DNA repair proteins – colon cancerLKB1 = ser/thr protein kinase 11 – GI tract polypsVHL = Von Hipple-Lindau – kidney cancer

LKB1 LKB1 kinasekinase activates AMPK & its family membersactivates AMPK & its family members


LST8

LST8

RaptorRaptor


Multiple Multiple mTORmTOR regulators are mutated in cancer regulators are mutated in cancer

PI3K activating mutations (p110PI3K activating mutations (p110αα))found in ~25% primary breast found in ~25% primary breast tumors. tumors.

Loss of PTEN (LOH) orLoss of PTEN (LOH) orpromoter promoter methylationmethylation--~30% breast tumors~30% breast tumors

~30% breast ~30% breast tumors have chromosomal tumors have chromosomal loss in 19p13.2loss in 19p13.2--13.313.3LKB1 locusLKB1 locus


The The RasRas/ERK & PI3K pathways /ERK & PI3K pathways are altered in most cancers. are altered in most cancers.


Concentrated on PI3K/PKB pathway Concentrated on PI3K/PKB pathway







Tumor cells appropriate a blood supply from the host tissue by Tumor cells appropriate a blood supply from the host tissue by stimulating stimulating neoangiogenesisneoangiogenesis;;this process is required for tumor growth. this process is required for tumor growth.

Tumors and Tumors and neoangiogenesisneoangiogenesis

RTKsRTKs of the VEGF family and their ligands are involvedof the VEGF family and their ligands are involvedin tumorin tumor--induced induced neoangiogenesisneoangiogenesis..

VEGF = vascular endothelial cell growth factorVEGF = vascular endothelial cell growth factor

Ongoing angiogenesis & the increase in Ongoing angiogenesis & the increase in microvesselmicrovessel densitydensityaids invasive tumor cells to disseminate through the bloodstreamaids invasive tumor cells to disseminate through the bloodstreamto distant to distant metastaticmetastatic sites. sites.

VEGF receptor/VEGF Network VEGF receptor/VEGF Network

Lymphangiogenesis is controlled by VEGF-C/VEGF-D & theirreceptor VEGFR-3 present on lymphatic endothelium.

Tumor-associated angiogenesis is controlled by VEGF-A & itsreceptor VEGFR-2 present on vascular endothelium.

TammelaTammela et al 2004 et al 2004 Cardio.ResCardio.Res 65:55065:550

Tumor Tumor neoangiogenesisneoangiogenesis, VEGF & HIF, VEGF & HIF--11

Tumor cells produce VEGF,Tumor cells produce VEGF,and/or stimulate neighboring cells in the and/or stimulate neighboring cells in the stromastroma to synthesis it.to synthesis it.

Hypoxia stabilizes the TF HIFHypoxia stabilizes the TF HIF--11α, α, the VEGFthe VEGF promoter has a promoter has a HIFHIF--11αα binding site. binding site.

VEGF expression is tightly controlled; in the tumor settingVEGF expression is tightly controlled; in the tumor settingmultiple pathways contribute to its multiple pathways contribute to its upregulationupregulation, an important, an importantone being HIFone being HIF--11αα. . HIFHIF--11αα = hypoxia inducible factor= hypoxia inducible factor

PouyssegurPouyssegur et al 2006 Nature 441: 437et al 2006 Nature 441: 437

The VHL Tumor SuppressorThe VHL Tumor Suppressor

GermlineGermline mutations in VHL are responsible for von mutations in VHL are responsible for von HippleHipple--LindauLindaudisease, characterized by a predisposition to disease, characterized by a predisposition to tumorigenesistumorigenesisin the CNS, pancreas & kidney. in the CNS, pancreas & kidney.

VHL is inactivated in ~80% of sporadic,VHL is inactivated in ~80% of sporadic,clearclear--cell renal cancer. cell renal cancer.

VHL targets HIFVHL targets HIF--11αα for oxygenfor oxygen--dependent dependent ubiquitinationubiquitination & & degradation. degradation.

KrekKrek 2000, Nature Cell 2000, Nature Cell BiolBiol

OxygenOxygen--dependent degradation domain (ODD)dependent degradation domain (ODD)

VCBVCB--Cul2 complexCul2 complex

VHL, VHL, ElonginElongin B & C B & C complexedcomplexed withwithCul2 Cul2 -- form the VCBform the VCB--Cul2 complexCul2 complexwhich targets Hifwhich targets Hif--11α α for oxygenfor oxygen--dependent degradation. dependent degradation.

E1 activates E1 activates ubiquitinubiquitin

Activated Activated ubiquitinubiquitin is transferredis transferredto the E2 to the E2 ubiquitinubiquitin--conjugating enzymeconjugating enzyme

The E3 The E3 ubiquitinubiquitin ligaseligase confersconfersspecificity in targeting specificity in targeting UbUb totoappropriate substrates appropriate substrates --promoting degradation promoting degradation

VHL is an E3 VHL is an E3 ubiquitinubiquitin ligaseligase

Under conditions of Under conditions of normoxianormoxiaHIFHIF--11αα is continually degraded. is continually degraded.

Pro residues in the ODD Pro residues in the ODD domain are domain are ––OHOH& Hif& Hif--1 is degraded . 1 is degraded .

Hypoxia blocks Hypoxia blocks ubub--mediated mediated HIFHIF--11αα degradation. degradation.

HIFHIF--11α α degradationdegradation

The tumor suppressor VHLThe tumor suppressor VHL

VHL mutations in kidney cancer promote stabilization ofVHL mutations in kidney cancer promote stabilization ofHIFHIF--11αα, an important regulator of , an important regulator of proangiogenicproangiogenic factors factors including VEGF. including VEGF.

Mutations in VHL mimic hypoxia Mutations in VHL mimic hypoxia ––

HIFHIF--11αα degradation is preventeddegradation is prevented

PouyssegurPouyssegur et al 2006 Nature 441: 437et al 2006 Nature 441: 437

HIFHIF--11α α controlcontrol

* To read more on how hypoxia impacts on * To read more on how hypoxia impacts on mTORmTOR see:see:J. J. PouyssegurPouyssegur et al 2006 Nature 441:437 et al 2006 Nature 441:437

Tumor angiogenesis Tumor angiogenesis -- a therapeutic target a therapeutic target

Therapeutics that target the vascular endothelial cells, Therapeutics that target the vascular endothelial cells, which supply tumors with oxygen & nutrients, are predictedwhich supply tumors with oxygen & nutrients, are predictedto spare normal tissue to spare normal tissue -- leading to hypoxia & tumor cell death. leading to hypoxia & tumor cell death.

In normal adults angiogenesis is basically arrested;In normal adults angiogenesis is basically arrested;tumors are very dependent upon tumors are very dependent upon neoangiogenesisneoangiogenesis..

Tumors also induce Tumors also induce lymphangiogenesislymphangiogenesis; the ; the lymphaticslymphatics carry carry tumor cells to the lymph nodes & to distant organs.tumor cells to the lymph nodes & to distant organs.

Compounds targeting the VEGFR/VEGF network are used Compounds targeting the VEGFR/VEGF network are used in cancer treatment. in cancer treatment.

PTK787 PTK787

Targeting the VEGFR/VEGF Network Targeting the VEGFR/VEGF Network

AntiAnti--VEGFVEGF

VEGFVEGF--AA

VEGF blocking antibodies

Bevacizumab/AvastinBevacizumab/Avastin: humanized : humanized mAbmAbtargeting human VEGFtargeting human VEGF--AA

VEGFR TK inhibitors

PTK787: PTK787: VEGFRVEGFR--1, 1, --2 & 2 & --3 3

ERBB Receptors & CancerERBB Receptors & Cancer

Specific alterations in ERBB receptor expression Specific alterations in ERBB receptor expression and/or activation have been found in many human and/or activation have been found in many human tumors.tumors.

Breast, ovarian, NSCLC, Breast, ovarian, NSCLC, glioblastomaglioblastoma, SCC head , SCC head & neck, colon, bladder, etc.& neck, colon, bladder, etc.

ERBB Receptors and CancerERBB Receptors and Cancer

Cancer patients whose tumors have ERBB receptorCancer patients whose tumors have ERBB receptoralterations tend to have a more aggressive diseasealterations tend to have a more aggressive diseasethat is associated with factors predicting a poorthat is associated with factors predicting a poorclinical outcome. clinical outcome.

ERBB receptors have been intensely pursued as ERBB receptors have been intensely pursued as therapeutic targets. therapeutic targets.

antibodiesantibodiessmall molecule tyrosine small molecule tyrosine kinasekinase inhibitors (inhibitors (TKIsTKIs))


PPPPP

PPPPP

PPPPP

PPPPP

MAPK

AKT

STAT

SRC

mTOR

PPPPPPPPPP

PPPPP

PPPPP

PPPPP

PPPPPPPPPP



PPPPPPPPPPPPPPP

PPPPP PPPPPPI3K



ERBB Receptors

X

Z


TKI

TKI



Trastuzumab(Herceptin)

Pertuzumab(Omnitarg)

Cetuximab(Erbitux)

Matuzumab

Panitumumab

ErbB2

ErbB2

EGFR

EGFR

EGFR

Genentech/Roche

Genentech

ImClone/Merck KGaABristol-Myers Squibb

Merck KGaA Phase II – NSCLC, gynecologic, pancreatic, Esophageal.

Abgenix

Approved ErbB2-overexpressing breast cancer; trials in combination with various drugs ongoing.

Phase II - ovarian, breast, prostate, NSCLC; inhibits ErbB2 dimerization; trials ongoing in low ErbB2expressors.

Approved CRC; trials in combination with various drugs ongoing- pancreatic, HNSCC & NSCLC.

Pivotal trial ongoing – 3rd

line CRC, other indications-RCC and NSCLC.

ErbBErbB targeted recombinant antibodies*targeted recombinant antibodies*

*humanized or *humanized or chimericchimeric

TargetTarget CompanyCompany

Hynes & Lane 2005 Nature Rev Cancer 5: 341Hynes & Lane 2005 Nature Rev Cancer 5: 341

ErbBErbB targeted targeted TKIsTKIs

TargetTarget CompanyCompany

Gefitinib(Iressa)

Erlotinib(Tarceva)

Lapatinib

AEE788

CI-1033

EKB-569

EXEL 7647/EXEL 0999

EGFR

EGFR

EGFR/ErbB2

EGFR/ErbB2/VEGFR

EGFR/ErbB2*

EGFR/ErbB2*

EGFR/ErbB2/KDR

AstraZeneca

Genentech/OSI Pharma

Approved 3rd line NSCLC, broad focus development.

GlaxoSmithKline

Novartis

Pfizer

Wyeth-Ayerst

EXELIXIS

Phase II - NSCLC

Approved 3rd line NSCLC, ongoing trials in other indications - HNSCC, GI & breast.

Phase III – Herceptin- & chemo-refractory breast cancer, other ongoing trials - refractory CRC

Phase I - First multi-function EGFR/ErbB2/VEGFR inhibitor; multiple potential Phase II indications.

Phase II – Breast, NSCLC

Phase I

*irreversible Hynes & Lane 2005 Nature Rev Cancer 5: 341Hynes & Lane 2005 Nature Rev Cancer 5: 341

ERBB Receptors and CancerERBB Receptors and Cancer

Understand mechanisms underlying activity of Understand mechanisms underlying activity of ERBB targeted inhibitors.ERBB targeted inhibitors.

How are patients chosen for treatment with a How are patients chosen for treatment with a specific inhibitor? specific inhibitor?

ERBB inhibitors in combination with other signal ERBB inhibitors in combination with other signal transduction inhibitors (transduction inhibitors (STIsSTIs).).

What mechanisms contribute to resistance?What mechanisms contribute to resistance?

EGFR EGFR kinasekinase domain mutations & NSCLCdomain mutations & NSCLC

In the initial clinical trials on In the initial clinical trials on gefitinibgefitinib & & erlotiniberlotinib,,most nonmost non--small cell lung cancer patients who respondedsmall cell lung cancer patients who respondedhad mutations in the had mutations in the kinasekinase domain of EGFR. domain of EGFR.

Lynch et al 2004 N Lynch et al 2004 N EnglEngl J Med 350 J Med 350 PaezPaez et al., 2004 Science 304et al., 2004 Science 304PaoPao et al 2004 Proc et al 2004 Proc NatlNatl AcadAcad SciSci 101101

First report on EGFR KD mutations.First report on EGFR KD mutations.

Clinical results suggested that KD mutations would Clinical results suggested that KD mutations would make a tumor more sensitive to make a tumor more sensitive to TKIsTKIs. .

Shigematsu & Gazdar 2006 Int J Cancer 118:257

Mutations target key structures around Mutations target key structures around the ATP binding cleft of the the ATP binding cleft of the kinasekinase domain domain

Mutations are predicted to keep the Mutations are predicted to keep the kinasekinasein the active conformation.in the active conformation.


EGFR EGFR kinasekinase domain mutations are domain mutations are oncogeniconcogenic

All EGFR KD mutations are All EGFR KD mutations are oncogeniconcogenic --mutant receptors transform* fibroblasts & epithelial cells.mutant receptors transform* fibroblasts & epithelial cells.((GreulichGreulich et al 2005 et al 2005 PLoSPLoS Medicine 2:e313)Medicine 2:e313)

(*soft agar growth, focus formation & tumor formation)(*soft agar growth, focus formation & tumor formation)

Frequency of Frequency of kinasekinase domain mutations domain mutations in EGFR/ErbB2 in NSCLC in EGFR/ErbB2 in NSCLC

5 studies exons 18-21 sequencedn = 1,108 - 350 mutations detected(31.6%)

EGFR EGFR

ErbB2ErbB2

3 studies sequenced KDn = 857 - 16 mutations detected(1.8%)


GermlineGermline mutation in EGFR mutation in EGFR kinasekinase domaindomain

A family with multiple cases of NSCLC associated with A family with multiple cases of NSCLC associated with germlinegermlinetransmission of an EGFR T790M mutation was described by D. Habertransmission of an EGFR T790M mutation was described by D. Haber& colleagues & colleagues (Bell et al 2005 December, Nature Genetics)(Bell et al 2005 December, Nature Genetics)

Gatekeeper residue in the ATP binding site

EGFR mutations & EGFR mutations & gefitinibgefitinib response in NSCLCresponse in NSCLC

6565--83% response rate for patients with mutant EGFR83% response rate for patients with mutant EGFR1010--14% 14% ““ WT EGFRWT EGFR

Why are the mutant receptors more sensitive to Why are the mutant receptors more sensitive to gefitinibgefitinib? ?

(Johnson & Janne 2005 JCO 23:6813)

Gazdar et al 2004 Trends Mol Med vol 10

Gefitinib/erlotinibGefitinib/erlotinib might bind the mutant might bind the mutant kinasekinase domain more readily domain more readily --In vitro evidence suggesting that mutant EGFR is more sensitive In vitro evidence suggesting that mutant EGFR is more sensitive to to TKIsTKIs

Predictions from EGFR Predictions from EGFR kinasekinase domain structure domain structure

Resistance to ERBB targeted Resistance to ERBB targeted TKIsTKIs

Resistance to Resistance to TKIsTKIs has emerged as a significant clinical problem, initiallyhas emerged as a significant clinical problem, initiallyin the context of CML & mutations in BCRin the context of CML & mutations in BCR--ABL, leading to ABL, leading to GleevecGleevec resistance. resistance. In clinical studies, T790M mutations were reported in cases of NIn clinical studies, T790M mutations were reported in cases of NSCLC thatSCLC thatrecur after initial TKI response. recur after initial TKI response.

The irreversible EGFR TKI CLThe irreversible EGFR TKI CL--387,785 blocks activity 387,785 blocks activity of receptors with T790M mutations. of receptors with T790M mutations.

TrastuzumabTrastuzumab & Breast Cancer& Breast Cancer--Questions that need to be addressed Questions that need to be addressed

How does How does tratuzumabtratuzumab function? function?

What contributes to What contributes to tratuzumabtratuzumab resistance / insensitivity? resistance / insensitivity?

% Cell Cycle Stage% Cell Cycle Stage

G1G1 SS G2/MG2/M

PBSPBS 6767 1212 2121

FRP5FRP5 6565 1616 2020

TrastuzumabTrastuzumab 9696 11 33

TrastuzumabTrastuzumab blocks proliferation of ErbB2blocks proliferation of ErbB2--ovexpressing breast cancer cellsovexpressing breast cancer cells

Lane et al 2000 MCB 20:3210Lane et al 2000 MCB 20:3210

This is accompanied by downThis is accompanied by down--regulation of pathway activity regulation of pathway activity ––PI3K/Akt. PI3K/Akt.

EGFR ErbB3 ErbB4Ferguson et al. (2003) Mol Cell Cho & Leahy (2002) Science Bouyain & Leahy (2005) PNAS

ErbB2Cho et al. (2003) Nature

ERBB receptor structures

In the absence of ligands EGFR, ErbB3 & ErbB4 are In the absence of ligands EGFR, ErbB3 & ErbB4 are ““closedclosed””..

ErbB2 has an extended structure.ErbB2 has an extended structure.

IIIIIIIV

Provided by D. LeahyProvided by D. Leahy

ErbB2 structure similar to ErbB2 structure similar to ligandligand--activated EGFRactivated EGFR

sErbB2sEGFR/EGF

EGF

IIIIIIIV


ErbB2

Cho et al. (2003) NatureCho et al. (2003) Nature

TrastuzumabTrastuzumab

ErbB2/trastuzumab structure has been solvedErbB2/trastuzumab structure has been solved

IIIIIIIV

TrastuzumabTrastuzumab binding binding neither blocks ErbB2 neither blocks ErbB2 dimerizationdimerization with the other with the other ligandligand activated ERBB receptors, activated ERBB receptors, nor does it prevent ErbB2 activationnor does it prevent ErbB2 activation. .


+BTC+HRG+EGF+PBS

93%

56% 61%

72% 60% 62%

57% 59%

+4D

5N

o tr

eat.

+BTC+HRG+EGF+PBS

93%

56% 61%

72% 60% 62%

57% 59%

+4D

5N

o tr

eat.

MotoyamaMotoyama et al., 2002 Cancer Res 62: 3151et al., 2002 Cancer Res 62: 3151

TrastuzumabTrastuzumab induced G1 block can be overcome by induced G1 block can be overcome by treatment with treatment with ErbBErbB ligandsligands

BT474 breast tumor cellsBT474 breast tumor cells

tras

tuzu

mab

tras

tuzu

mab

Cont

rol

Cont

rol

TrastuzumabTrastuzumab ResistanceResistance

Tumors might escape from trastuzumab if ERBB ligandsare present in high concentrations in the tumor environment.

One potential mechanism of trastuzumab resistance.

Provides a rationale for developing pan-ERBB inhibitors.


PPPPP

PPPPP

PPPPP

PPPPP

MAPK

AKT

STAT

SRC

mTOR

PPPPPPPPPP

PPPPP

PPPPP

PPPPP

PPPPPPPPPP



PPPPPPPPPPPPPPP

PPPPP PPPPPPI3K



ERBB Receptors

X

Z


TKI

TKI



Mutations in signaling proteins downstream of ERBB receptorsmay play a role in sensitivity/resistance to ERBB targeted inhibitors.

Protein Production

GeneTranscription

Cell GrowthProliferation

Integrins

ILK

4E-BP1

eIF-4E P 70 S6k

PTEN

TSC1/2

mTOR

PI3K

AKT/PKB

FKBP12

Growth Factors

Rationale for combination of Rationale for combination of mTORmTOR + ERBB inhibitors+ ERBB inhibitorsDeregulated PI3K/Akt signalingDeregulated PI3K/Akt signalingactivates activates mTORmTOR, but pathway mutations , but pathway mutations can uncouple can uncouple mTORmTOR from the receptor.from the receptor.

Low PTEN levelsLow PTEN levels--implicated in resistance to implicated in resistance to TrastuzumabTrastuzumab..(Nagata et al., 2004 Cancer Cell)(Nagata et al., 2004 Cancer Cell)

Loss of negative regulators Loss of negative regulators --PTEN PTEN –– often found in often found in Breast cancer.Breast cancer.

PIK3CAPIK3CA mutations have beenmutations have beenfound in ~ 25% breast cancers.found in ~ 25% breast cancers.

Protein Production

GeneTranscription

Cell GrowthProliferation

Integrins

ILK

4E-BP1

eIF-4E P 70 S6k

PTEN

TSC1/2

mTOR

PI3K

AKT/PKB

FKBP12

Growth Factors

ErbBErbB inhibitor AEE788 inhibitor AEE788 ((TraxlerTraxler et al., 2004 Cancer Res 64)et al., 2004 Cancer Res 64)

RAD001

RAD001 binds the RAD001 binds the immunophilinimmunophilin FKBP12 FKBP12 & blocks & blocks mTORmTOR activityactivity((BoulayBoulay et al., 2004 Cancer Res 64)et al., 2004 Cancer Res 64)

Rationale for combination of Rationale for combination of mTORmTOR + + ErbBErbB inhibitorsinhibitors

Despite the multiple alterations in a cancer cell Despite the multiple alterations in a cancer cell --

Our increasing knowledge of the Our increasing knowledge of the mechanisms & pathways underlying mechanisms & pathways underlying cancer development has led to the cancer development has led to the development of new therapeutic development of new therapeutic strategies ! strategies !

Major goals for the future: Major goals for the future: -- choose the patient population to treat with a targeted inhibitochoose the patient population to treat with a targeted inhibitorr-- choose the optimal combination of inhibitorschoose the optimal combination of inhibitors-- understand mechanisms of resistance understand mechanisms of resistance


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Disturbances of Growth Control- · bind Pro-rich regions ... 2012-2020 GIST patient w abdominal mets before Glivec treatment 48 hr after Glivec 8 days after Glivec Positron Emission