Phosphoproteomicsand Cancer

Scott A. Gerber, PhDDepartments of Genetics and Biochemistry

Norris Cotton Cancer CenterGeisel School of Medicine at Dartmouth

CDK1Plk1

Aurora ACDK1Plk1

Aurora ACDK1Plk1

Aurora A/B

CDK1Plk1

Aurora B

Plk1Aurora B

PP2A/PP1

PP2APP1

PP1 Prophase Prometaphase

Metaphase

AnaphaseTelophase

Cytokinesis

Interphase

Kinases, cell division and cancer

multipolarspindle

chromosomemissegregation

aberrant cytokinesis

spindle defects

Prophase Prometaphase

Metaphase

AnaphaseTelophase

Cytokinesis

Interphase

Kinases, cell division and cancer

Metaphase

AnaphaseTelophase

Cytokinesis

Prophase Prometaphase

Interphase chromosome instability

&aneuploidy

multipolarspindle

chromosomemissegregation

aberrant cytokinesis

spindle defects

Kinases, cell division and cancer

Phosphoproteomics and cancer: opportunities, challenges & progress

adapted from Pao and Girard, Lancet 2011

Genomics-based targeted treatments:

•EGFR (~10%) – gefitinib/erlotinib

•ALK (~4%)- crizotinib

•RAS/RAF (~28%) – MEK inhibitors

Phosphoproteomics and cancer: opportunities, challenges & progress

*http://cancer.sanger.ac.uk/cancergenome/projects/cosmic,La Locano 2011, J Trans Med; Wolf 1997, Oncogene

PLK1 mRNA expressionN T N T N T

N – normal tissueT – tumor tissue

8,300 tumors

61 (0.7 %) mutations

COSMIC*

9,200 tumors

35 (0.4 %) mutations

Drug(s)

BI6727 (volasertib)Phase III

MLN8237 (alisertib)Phase III

Plk1AurkA

Plk1AurkA

Plk1

Schwanhäusser et al. 2011, Nature; Lundberg et al. 2010, Mol. Sys. Biol

U-2OS A-432 U-251MG

NIH3T3

Phosphoproteomics and cancer: opportunities, challenges & progress

α-Plk1

H23H1650

H1838H1975

H2170H1395

H522

18.0 19.0 20.0 21.0 22.0Time (min)

Ab

un

dan

ce

AQUA

endogenous

HINPVAASLIQK

0 0.5 10

0.5

1

relative Plk1 mRNA abundance

rela

tive

Plk1

pro

tein

abu

ndan

ce

r2 = 0.04

Polo-like kinase 1 (Plk1): protein vs mRNA vs chemosensitivity

Kettenbach & Gerber, Nature Protocols (2011)

Phosphoproteomics and cancer: opportunities, challenges & progress

0 0.5 10

0.5

1

relative Plk1 protein abundance

rela

tive

LD50

Plk

1 in

hibi

tor r2 = 0.17

Phosphoproteomics and cancer: opportunities, challenges & progress

P

ATP ADP

P

kinase

phosphatase

cdk1

PP1 PP1

P

“active”

Cell cycle progression

G1 S G2 M

Cdk1 activity

PP1 activity

G1

Tumor phosphoproteomes represent the balance of opposing activities

PP

P

P

AB C

PP

P

P

A’B C’

OH

Biol

ogy

Condition 1 Condition 2

Stable Isotope Labeling by Amino acids in Cell culture

anti-A antibody, etc.

Expe

rimen

t 12C & 14N Lys & Arg(light)

13C & 15N Lys & Arg(heavy)

each tryptic peptideends in Lys or Arg & can be quantified

- mix conditions- lyse

- IP A/A’-SDS-PAGE & digest

mixing cells allowsmulti-step, subcellularfractionation &accurate quantitation

Info

rmati

on

100%

50%

m/z

B

C’C

A’A

P

AP

A’

A’OH

P

A’PA

AOH

condition (1): lightcondition (2): heavy

Quantities are ratios:e.g. heavy / light

Ong et al., Molecular & Cellular Proteomics 2002

Quantitative Proteomics: SILAC

Spike-in SILAC analysis of human lung cancer tumors

Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

Spike-in SILAC analysis of human lung cancer tumors

Two non-small cell lung cancer (NSCLC) tumors

Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

Spike-in SILAC analysis of human lung cancer tumors

Motif-X – http://motif-x.med.harvard.edu/

Target discovery

Dimensionality reduction

Motif-based biomarkers

Spike-in SILAC analysis of human lung cancer tumors

Spike-in SILAC analysis of human lung cancer tumors

Kettenbach et al., Science Signaling (2011); Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

00.10.20.30.40.50.60.7

0 20 40 60

TiO2 LC-MS/MSinhibited

13C15N

control12C14N

+

combinelyse

trypsin digestion SCX

chromatography

phosphopeptide enrichment

N

HN

OHN N

N

N

N O

O

N

HN

OHN N

N

N

N O

O

Plk1 inhibitor

> 700 Plk1 candidate substrate phosphorylation sites

previously:

in these lung tumors:

Kinase-specific “substrate-omes” may be reflective of in vivo activity

Spike-in SILAC analysis of human lung cancer tumors

Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

What about protein abundance differences?

Tumor phosphoproteomes contain greaterdynamic information than proteomes

Translational phosphoproteomics: Status

Currently moving forward with the analysis of 40 NSCLC lung tumors for phosphoproteomic analysis

• Study population• Subjects undergoing thoracic surgery for presumed lung

cancer

• Determine if significant correlation exists between Aurora A and/or Plk1 substrates and progression-free survival

• Correlate global phosphoproteomics patterns with disease-free survival, time to disease recurrence, lung cancer-specific survival and overall survival

• New instrumentation affords deeper coverage• New instrumentation enables multiplexed analyses

AcknowledgementsThe Gerber Labproteomics.dartmouth.edu Dr. Lilian KabecheDr. Devin Schweppe (past)Jason GilmoreJeffrey MilloySierra CullatiKatelyn CassidyAndrew GrassettiMark Adamo

The Kettenbach Labwww.kettenbachlab.org Dr. Arminja KettenbachAdam PetroneScott RusinKate Schlosser

NCCC Thoracic OncologyDr. James RigasDr. Konstantin Dragnev

FundingAmerican Cancer SocietyNIH P20-GM103413

R01-CA155260S10-OD016212

Cambridge Isotopes ThermoFisher Scientific GL Sciences