Using 13C and 15N Isotopomer Metabolic Flux via Glucose and Glutamine to Understand Cancer’s

Metabolic Dependencies by SRM-LC-MS/MS

John M. Asara, Ph.D.

Beth Israel Deaconess Medical Center Harvard Medical School

Boston, MA

ASMS 2013 - Minneapolis

Cancer's insatiable appetite. Locasale et. al. Nat. Biotechnol., 2009

It’s more than just kinase activity and genetic defects in cancer

Signaling Network

Metabolism

Cell growth and proliferation (Cancer)

‘Warburg Effect states that glucose is taken up at a high rate and converted to lactate by cancer cells’

Engelman et. al., Nat. Medicine, 2008

FDG – PET scan (labeled glucose)

+TKI

Lung tumor

•We can use this to our advantage when tracing labeled carbon through cancers

13C-glutamine

13C-glucose

Simplified to Central Carbon Metabolism ~200 metabolites for 13C/15N Tracing

Map of the Human Metabolome

AB/SCIEX 5500 QTRAP

Selected Reaction Monitoring (SRM) ~300 transitions (258 unique metabolites -12C & 13C)

Metabolite Selection

Fragment ion Selection

Fragmentation

4.6mm x 15cm

400 µL/min pH=9.0, NH4

+

Amide XBridge HILIC - 1 column

Platform for Targeted Endogenous Polar Metabolite Profiling

+/- switching

PCA analysis MarkerView

Metaboanalyst.ca Clustering (MatLab, Metaboanalyst.ca) KEGG pathway mapping

Extract metabolites with 80% methanol From cells, tumor tissue, fluids, etc.

MultiQuant v2.0 Peak Area integration software

Cancer cells

Mean R2 = 0.978

Mean CV= 0.12

FWHH = ~9 seconds

Cycle time = 1.67 sec

3-4 msec dwell

10-14 points per peaks

Yuan, et. al., Nature Protocols, 2012.

Steady-State Metabolic Flux Analysis: measure the destination of the

labeled carbon atoms through glycolysis and related pathways

13C6-labeled glucose

RTK

Cell growth/

proliferation

of cancer

“SILAC” version for metabolomics

13C5-labeled glutamine

Glucose – Feeds glycolysis

Glutamine – Feeds TCA cycle

Cambridge Isotope Laboratories (CIL) Cancer cell

15N-labeled glutamine

-KG

Tracking Glutamine Metabolism by Isotopomer Steady-State Flux

Malate

Isocitrate

Fumarate

OAA

Citrate

Succinate

Glu

Labeled

Glutamine

Ac-CoA

Unlabeled

Glucose

Pyruvate

-KG Isotopomers

Fully glucose-

derived

Fully glutamine-

derived

Degrees of

glucose carbon

incorporation

into glutamine-

derived αKG

0

0.1

0.2

0.3

0.4

0.5

13C0 13C2 13C3 13C4 13C5

% 1

3C

-lab

ele

d M

eta

bo

lite

-KG Isotopomer Analysis in Gln-addicted Pancreatic Cancer

Carbon 12

Carbon 13

SRM Transitions

Example of 13C Labeling in Cells Showing that Glutamine Predominantly Fuels the TCA Cycle

TCA Cycle 13C-glutamine

13C-glucose

13C Glutamine and 13C Glucose Labeling in H929 Multiple Myeloma cells

13C-Glucose 13C-Glutamine

Top 75 featuresWard Heat Map

Top 75 featuresWard Heat Map

293T

H929

293T

H929Multiple Myeloma

Control

Multiple Myeloma: a cancer of the plasma cells in bone marrow

• Hypercalcemia (bone loss) • Lesions / Fractures • Abnormal blood levels • Low immunity (excess monoclonal IgG)

-0.4

-0.2

0

0.2

0.4

0.6

0.8

0h 24h 48h 72h 96hV

iab

ility

[%

]

H929 Viability Assay

Glucose

Glutamine

968 (Glutaminase inhibitor, TCA inhibitor) 2DG (Glucose competitor, Glycolysis inhibitor) 6AN (G6PD inhibitor, PPP inhibitor)

H929 Multiple Myeloma Cells Treated with Metabolic Inhibitors and Targets can be checked with 13C Glucose Tracing

Normal growth

968

TCA inhibitor

2DG

Glycolysis inhibitor

•H929 myeloma cells are more highly dependent upon glucose than glutamine for growth via PPP and glycolysis

6AN

PPP inhibitor (critical)

H929 Cells Respond to Kinase Inhibition through Nucleotide Accumulation – Transcription Inhibition and RNA Degradation

ThuP17 Poster #296, Susanne Breitkopf

Biological Model for H929 Polar Metabolomics

Phosphoproteomics

Growth and Proliferation

Ben-Sahra et. al., Science, 2013

S6K

TSC1/TSC2

Rapamycin

The TSC1/TSC2 – mTOR Pathway is a Metabolic Switch for Cell Growth and Proliferation

mTOR

S1859 PO4

Pyrimidine synthesis pathway

Unlabeled Polar Profiling (selected from 225 metabolites)

U-13C6 Glucose Intraperitoneal (IP) Injection experiments: 13C-glucose solution (2g/kg; fasted O/N). 30-60 min later, the mice were sac’d and tumor and other organs were harvested.

U-13C Glucose Jugular Bolus : Infusion experiments: 400mg/kg 13C glucose in 0.2 mL saline : 12 mg/kg/min at 150 µL/hr for 1.5 hr. tumor, liver

and serum were harvested. Mice died after 30 min.! – No longer do this.

Two methods of Injecting 13C into mice: Which method works best?

U13C6-labeled glucose

13

13 13

1313

13

Extract metabolites for LC-MS/MS

In Vivo Labeling of 13C-Glucose

13C

Tracking blood glucose and early sacrifice improves 13C labeling

-0.05

0.05

0.15

0.25

0.35

0.45

0.55

0.65

%U

13-g

luco

se la

bel G6P

F6P

F1,6BP

G3P

3PG

PEP

lactate

0

0.1

0.2

0.3

0.4

0.5

0.6

%U

13-g

luco

se l

ab

el

G6P

F6P

FBP

G3P

3PG

2,3-BPG

PEP

lactate

30-60 min sacrifice, monitor blood glucose (low level=high glucose uptake)

2 hr sacrifice, low glucose uptake, no blood monitor

Low 13C labeling

High 13C labeling

Glycolysis

Glycolysis

In Vivo Labeling of mice

LC-MS/MS (SRM)

Test blood glucose/sac mice

Use of Glucose Tolerance Test to Monitor Optimal Sac time of mice

Labeling with 13C can be achieved to ~50-60% incorporation

Mouse Sick Tumor Dox Weight

(g)

Sac’d

(min)

Blood

[Glucose]

(mg/dL)

#1 Yes Yes, huge No 29 30 113

#2 No No No 34 60 315

#3 No No Yes 44 60 212

#4 No Yes, very small Yes 29 30 193

•Large tumors take up glucose at a rapid rate, lowering blood glucose levels

0

0.1

0.2

0.3

0.4

0.5

0.6

%U

13-g

luc

os

e l

ab

el

G6P

F6P

FBP

G3P

3PG

2,3-BPG

PEP

lactate

Glycolysis

* * * *

Significantly (P < 0.05) differentially regulated metabolites in

labeled tumors upon PI3K Inhibitor treatment

glucosamine_12+13 6PG_12+13 6PG_13 guanosine_13C Ga3P_12+13 aKG_12 Ser_13 OAA_13 Asp_13 fumarate_13 homocysteine_12 inosine_12 orotate_12 (Iso)citrate_12 Carnitine_12 taurine_12+13 NAD+_12 fumarate_12+13 RP_13 Gln_13 UDP-Glc_13

NB

#1

N

B#

2

NB

#3

N

B#

4

NB

#5

N

B#

6

NB

#7

.1

NB

#7

.2

NB

#8

T

#1

.1

T#

1.2

T

#2

T

#3

.1

T#

3.2

T

#4

T

#5

.1

T#

5.2

T

#6

T

#7

T

#8

Vehicle BYL P < 0.05 in tumor 13C-U6-Glucose

PI3K Inhibitor BYL Abrogates Flux Through Central Metabolism in 13C-Glucose Labeled Mouse Tumors

Ward & Thompson, Cancer Cell, 2012

No change

•Targeted MS can be used successfully track the fate of 13C or 15N labeled molecules in cells and in vivo •Tracking the activated or altered metabolic pathways in cancers is important for recognizing cancer cell types that have a growth survival benefit •Understanding these altered metabolic pathways is important for determining how we can intervene to block these pathways with “smart drugs”

Conclusions

Acknowledgements

Beth Israel Deaconess Medical Center / Harvard Medical School

Costas Lyssiotis – 13C labeling/SRM methods Susanne Breitkopf – 13C labeling/mass spec Min Yuan – mass spec Gary Bellinger –in vivo mouse experiments Lewis Cantley

Funding

National Institutes of Health Dana-Farber Harvard Cancer Center BIDMC