klarisa rikova, benjamin hall understanding of differing ...klarisa rikova cell signaling...

Wehavedevelopedauniqueproteomicapproachtoeluci-datechangesinsignalingpathwaysupondrugtreatmentinRTKdrivencelllines.

OurdatasuggestthatALKactivatesdifferentpathwaysinALKdrivencelllinesincomparisontocelllinesdriv-enbyEGFR/Met.

ThepresenceofEGFRasaparallelpathwayinH2228cellscouldexplainthedifferingsensitivityobservedtotheALKinhibitorcrizotinib.

GeldanamycininhibitedALKandotherRTKsinacrizotinib-sensitivecellline(H3122)andhadnoeffectsonRTKsinacrizotinib-insensitivecellline(H2228).

TherapywithALKinhibitorsaloneisonlyeffectiveinasubsetofNSCLCALK-positivepatientswithEML4-ALKfusions.IncasesofresistancetoALKinhibitors,combinationtherapymaybedesirable.

Summary

Contact InformationKlarisa Rikova

Cell Signaling Technology, Inc. Tel: (978) 867-2330 Email: [email protected]

Understanding of Differing Sensitivity in EML4-ALK NSCLC Patients to Crizotinib and GeldanamycinKlarisa riKova, Benjamin Hall, anthony Possemato, Keri mroszczyK, Kimberly a. lee, Joan macneill, Jian min ren, ailan Guo, Daniel mulhern, yi WanG, sean a. beausoleil, michael J. comb

www.cellsignal.com Cell Signaling Technology® and PhosphoScan® are registered trademarks of Cell Signaling Technology, Inc. / LTQ® and Orbitrap® are registered trademarks of Thermo Fisher Scientific Inc. / Sep-Pak® is a trademark of Waters Corporation.

Non-smallcelllungcancer(NSCLC)isthemostcommonformoflungcancer,afflictingnearly200,000

peopleintheUnitedStateseachyear.AbnormalALKisfoundinabout5%ofNSCLCcases,meaning

morethan5,000newpatientscouldbenefitfromthetyrosinekinaseinhibitor(TKI)crizotinib.

However,notallpatientsbenefitfromsuchtreatment,withtheclinicalresponsetocrizotinibdiffering

amongpatientswhoharborthesamemolecularabnormality.Similarly,patientswithALKfusion

proteinshaveshownvaryingsensitivitytotheHSP90inhibitorgeldanamycininpreclinicalstudies.

WehavechosentwoNSCLCcelllines,H3122andH2228,asamodeltoaddressthesequestions.

BothcelllinesharborEML4-ALKfusionswithdifferingsensitivitytotheinhibitorscrizotiniband

geldanamycin.WeusedtheestablishedmethodofTMTpeptidelabelingcoupledwithserialpeptide

immunoprecipitation.ToobservetheeffectsofcrizotinibandgeldanamycinonALKsensitiveH3122

andnon-sensitiveH2228NSCLCcelllinesquantitativeanalysiswasperformed.Phosphotyrosine,

acetylation,methylation,ATM/ATRsubstrate(s/tQ),Akt/AMPKsubsAGC/CAMK/STE,andMAPK

familykinasemotifantibodieswereusedforimmunoprecipitationallowingustocharacterizeand

quantifyposttranslationalmodificationsbeforeandaftertreatment.

Inthisstudy,weidentifyextensivesignalingnetworksdownstreamofALKacrossmultiplespaces,

includingphosphorylation,acetylation,andmethylation.Thesedifferencesmaybeclinicallysig-

nificantandhighlightthepossibilitythatALKinhibitorsalonemayonlybeeffectiveinasubsetof

NSCLCALK-positivepatientswithEML4-ALKinversion.

Abstract

Table 1. 2mg of each sample was labeled using TMT reagents with 100ug used for total proteome analysis and the rest used to profile PTM spaces. Labeled samples were then combined and subjected to sequential immunoprecipitations using motif antibodies for PTMs and in parallel basic reverse phase (bRP) chromatography was performed to profile the full proteome. Samples were purified over Sep-Pak® C18 columns before being processed by LC-MS/MS/MS analysis. Data generated from the MS analysis were processed through several modules in CORE, ending with quantitated data.

Table 2. Motif antibodies chosen for PhosphoScan® profile (MS/MS).

Figure 1. Experimental flowchart. Cell lysates were prepared from RTK inhibitor treated and untreated cell lines, proteins were digested with trypsin, peptides were TMT-labeled, and samples were mixed for 6-plex analysis. The peptide mixtures were serially fractionated through immunoprecipitation with antibodies specific for phosphotyrosine, ATM/ATR substrate motif, AGC/CAMK/STE family kinase motif, acetyl-lysine, and methyl-arginine, though any motif-recognizing antibody could be used with this protocol. The enriched peptides from each immuno-precipitation were analyzed by LCMS/MS using an LTQ® Orbitrap® Elite System, with quantification of the TMT labels enabled by HCD fragmentation.

Figure 2. RTK driven cell lines profiled through the following spaces: phosphotyrosine, phosphoserine/phosphothreonine, acetylation, methylation.

Figure 3. Number of proteins inhibited in different PTM classes (phosphotyrosine, ATM/ ATR substrate phosphorylation, AGC/CAMK/STE kinase family motif phosphorylation, AKT/AMPK, lysine acetylation, arginine methylation, lysine methylation) by greater than 3.8 fold upon TKI treatment.

Sample Channel

H3255 Control 126

H3255 Control 127

H3255 1 hr Iressa 128




MKN 45 Control Starved 126

MKN 45 Control Starved 127

MKN 45 1 hr Crizotinib 128




Sample Channel

H2228 Control Starved 126

H2228 Control Unstarved 127

H2228 1hr Crizotinib 128


H2228 15hr Geldanamycin 130


H3122 Control Starved 126

H3122 Control Unstarved 127





Ab Target Clone # Motif

pY-1000 D1G10/D2D1 Phospho Tyr

AGC/PSDAkt substrateAMPKATM/ATR substrateATM/ATR substrate

D3E5/D8D9/D4E2/D8B11100B7D72H3/D78G9D23/D69D14/D86

AGC/CAMK/STE kinase acitivation loopRXX(s/t)LXRXXT(S) (s/t)Q(s/t)Q

Ac-KK-MeR-MeEpigenetic Regulator Mix

D10G3/11D/16ED4P3J, D3Z9J, D8R1CMe-R4-100-

Acetyl lysineK-MeR-Me-

Ubiquitin Library D4A7A10 kGG

Cell Line

Control

Control

1 hr

Proteolytic Digestion

3 hr

6 hr

24 hr TMT Labeling

Labeled PeptidesMix Samples

Methylation

IP Methyl-arginine Peptides

Acetylation

IP Acetyl-lysinePeptides

Ser/Thr Kinases

IP AGC KinasesMotif Peptides

DNA Damage

IP ATM/ATRSubstrate Peptides

Tyr Kinases

IP Phospho TyrosinePeptides

LCMS/MS Analysis LCMS/MS Analysis LCMS/MS Analysis LCMS/MS Analysis LCMS/MS Analysis

TMT Peptide Quant TMT Peptide Quant TMT Peptide Quant TMT Peptide Quant TMT Peptide Quant

EGFR

Alk

Met

Methylases,Demethylases

Tyrosine Space

Ser/Thr Space

Acetylation

Methylation

Tyrosine Kinases

Serine Kinases

Acetylases, HDACs

Alk

EML4

H2228 (EML4-ALK)non-sensitive

Met

AXL

EphA2IGF1R

MERTK

EGFR

CIT

PRKAA1CDK1MAPK1MAPK10MAPK3MAPK9

PBK

PEAK1SGK223A

GC

CAM

K

CMGC

Othe

r

BCR

Atyp

ical

TK

Crizotinib


MAPK3SIK3

CDK1 MAP4K4STK39CA

MK

CMGC ST

E

Geldanamycin

H3122 (EML4-ALK)sensitive

EphA2 Met

CIT

PRKAA1BCR PBK

GSG2

SGK223

CAM

K

CDK1MAPK1MAPK10MAPK3

CMGC

Atyp

ical

Othe

r

TK

Crizotinib

AGC


Met

MetEGFR

EphA2

IGF1R

Geldanamycin

CITPKN1PKN2PRKCDPRKCEPRKCGPRKCIPRKCZSTK38L

ATRMTOR

CAMK1DCAMK2GMAPKAPK3MARK1MARK2MARK3PRKD2PRKD3SIK3

NEK9PLK1WNK1WNK2

MAP3K4MAP4K4STK10STK24STK39TAOK3

ARAFKSR1

PTK2PKM2TYK2

Atyp

ical

CAM

K

Othe

r

STE

TKL

TK

AGC

CDK1CDK11ACDK5MAPK14MAPK6

CMGC

Alk

Alk

EML4

Alk

Alk

EML4

Alk

Alk

EML4

Possible Secondary Driver


Met

AXL

EphA2IGF1R

MERTK

EGFR

CIT


PBK

PEAK1SGK223A

GC

CAM

K

CMGC

Othe

r

BCR

Atyp

ical

TK

Crizotinib


MAPK3SIK3

CDK1 MAP4K4STK39CA

MK

CMGC ST

E

Geldanamycin


EphA2 Met

CIT

PRKAA1BCR PBK

GSG2

SGK223

CAM

K


CMGC

Atyp

ical

Othe

r

TK

Crizotinib

AGC


Met

MetEGFR

EphA2

IGF1R

Geldanamycin


ATRMTOR


NEK9PLK1WNK1WNK2


ARAFKSR1

PTK2PKM2TYK2

Atyp

ical

CAM

K

Othe

r

STE

TKL

TK

AGC


CMGC

Alk

Alk

EML4

Alk

Alk

EML4

Alk

Alk

EML4


Figure 4. Kinases inhibited by greater than 3.8 fold change upon treatment with HSP90 inhibitor geldanamycin after 24hr treatment.


Met

AXL

EphA2IGF1R

MERTK

EGFR

CIT


PBK

PEAK1SGK223A

GC

CAM

K

CMGC

Othe

r

BCR

Atyp

ical

TK

Crizotinib


MAPK3SIK3

CDK1 MAP4K4STK39CA

MK

CMGC ST

E

Geldanamycin


EphA2 Met

CIT

PRKAA1BCR PBK

GSG2

SGK223

CAM

K


CMGC

Atyp

ical

Othe

r

TK

Crizotinib

AGC


Met

MetEGFR

EphA2

IGF1R

Geldanamycin


ATRMTOR


NEK9PLK1WNK1WNK2


ARAFKSR1

PTK2PKM2TYK2

Atyp

ical

CAM

K

Othe

r

STE

TKL

TK

AGC


CMGC

Alk

Alk

EML4

Alk

Alk

EML4

Alk

Alk

EML4



Met

AXL

EphA2IGF1R

MERTK

EGFR

CIT


PBK

PEAK1SGK223A

GC

CAM

K

CMGC

Othe

r

BCR

Atyp

ical

TK

Crizotinib


MAPK3SIK3

CDK1 MAP4K4STK39CA

MK

CMGC ST

E

Geldanamycin


EphA2 Met

CIT

PRKAA1BCR PBK

GSG2

SGK223

CAM

K


CMGC

Atyp

ical

Othe

r

TK

Crizotinib

AGC


Met

MetEGFR

EphA2

IGF1R

Geldanamycin


ATRMTOR


NEK9PLK1WNK1WNK2


ARAFKSR1

PTK2PKM2TYK2

Atyp

ical

CAM

K

Othe

r

STE

TKL

TK

AGC


CMGC

Alk

Alk

EML4

Alk

Alk

EML4

Alk

Alk

EML4


Figure 5. MTT assay showing sensitivity of H2228 to crizotinib vs. crizotinib combined with iressa.

Figure 7. Sum of fold change above 3.8 in RTK driven cell lines upon treatment with TKIs by protein type (transcriptional regulation, translation, protein kinsases, cytoskeletal/adhesion).

100

90

80

70

% o

f Con

trol

60

50

40

30

0 0.01 0.05 0.1

Drug Conc. (µM)

Alk Inhibitor, Crizotinib

Alk+EGFR Inhibitor (Iressa)

0.5 1 5 10

Inhibition of Kinase Signaling

H22280

200

400

600

800

1000

1200

H3122 H3255 MKN45

Protein Kinase, Ser/Thr

Protein Kinase, Tyr

Sum

of F

old

Chan

ge

Sum

of F

old

Chan

ge

Sum

of F

old

Chan

ge

Inhibition of Transcription/Translation Signaling

H22280

2000

1000

3000

4000

H3122 H3255 MKN45

Translation

Transcription

Inhibition of Adhesion/Cytoskeletal Signaling

H22280

6000

4000

2000

8000

10000

H3122 H3255 MKN45

Inhibition of Kinase Signaling

H22280

200

400

600

800

1000

1200

H3122 H3255 MKN45

Protein Kinase, Ser/Thr

Protein Kinase, Tyr

Sum

of F

old

Chan

ge

Sum

of F

old

Chan

ge

Sum

of F

old

Chan

ge

Inhibition of Transcription/Translation Signaling

H22280

2000

1000

3000

4000

H3122 H3255 MKN45

Translation

Transcription

Inhibition of Adhesion/Cytoskeletal Signaling

H22280

6000

4000

2000

8000

10000

H3122 H3255 MKN45

Figure 6. Kinases inhibited by greater than 3.8 fold change upon treatment with ALK inhibitor crizotinib after 1hr treatment.

klarisa rikova, benjamin hall understanding of differing ...klarisa rikova cell signaling...

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