A Metabolic Approach to Sarcoma Therapy

Brian A. Van Tine, M.D., Ph.D.Assistant Professor of MedicineSarcoma Program DirectorSWT Tower 731Washington University in St. Louis660 S. Euclid AvenueCampus Box 8007St. Louis, MO 63110Phone: 314-747-8475Pager:  314-508-4212 FAX:    314-362-7086

Disclosures

• Caris• DFINE• Polaris• AB Science• GSK• Novartis

Osteosarcoma

Kobayashi et. al. Mol Cancer Ther 2010;9:535-544

ASS1

• The protein encoded by this gene catalyzes the penultimate step of the arginine biosynthetic pathway.

• There are approximately 10 to 14 copies of this gene, the only functional copy is on chromosome 9.

• Mutations in ASS1 cause citrullinemia.

Soft tissue tumors Malignant Peripheral Nerve Sheath Tumor (NF1 Related) 3/44 Leiomyoma 0/20Malignant Peripheral Nerve Sheath Tumor (Non-NF1) 3/31 Myxofibrosarcoma (Myxoid MFH) 0/7Neurofibroma 0/19 Well-Differentiated Liposarcoma 0/6Plexiform Neurofibroma 0/24 Dedifferentiated Liposarcoma 1/9Diffuse-Type Neurofibroma 0/11 Myxoid Liposarcoma 10/12Cellular Schwannoma 0/7 Pleomorphic Liposarcoma 1/3 Clear Cell Sarcoma 0/7 Desmoid Fibromatosis 0/23Desmoplastic Melanoma 1/10 Dermatofibrosarcoma Protuberans 1/6 Fibrosarcomatous Dermatofibrosarcoma Protuberans 0/9 Desmoplastic Small Round Cell Tumor 1/6 Perineurioma 2/4 Endometrial Stromal Sarcoma 0/7Schwannoma 0/36 Epithelioid Sarcoma 2/3 Synovial Sarcoma 14/36 Low-grade Fibromyxoid Sarcoma 1/3Gastrointestinal Stromal Tumor 3/95 Epithelioid Hemangioendothelioma 0/2Sarcoma, NOS/Malignant Fibrous Histiocytoma 7/60 Angiosarcoma 0/5Embryonal Rhabomdyosarcoma 1/3 Extraskeletal Myxoid Chondrosarcoma 5/7 Alveolar Rhabomdyosarcoma 0/2 Nodular Fasciitis 0/6Pleomorphic Rhabomdyosarcoma 2/8Solitary Fibrous Tumor/Hemangiopericytoma 16/19Tenosynovial Giant Cell Tumor 2/34 Bone TumorsAngiomyolipoma 3/8 Giant Cell Tumor of Bone 0/7Glomus Tumor 1/5 Fibrous Dysplasia 0/9Granular Cell Tumor 0/4 Non-Ossifying Fibroma 1/6Myxoma 2/5 Osteosarcoma 3/10Leiomyosarcoma 8/56 Ewing Sarcoma/PNET 1/7

H&E ASS1 3+ H&E ASS1 0

ASS1 Immunohistochemistry on 701 Soft Tissue Tumors

MPNST ASS+ MPNST ASS -

619/701

88.3%-

CitrullineCarbamylPhosphate

N-acetylglutamateCPSI

HCO3 + NH4 + 2ATP

Argininosuccinate

ASS

Aspartate

ArginineFumarate

ASL

Ornithine

Urea

ARG

OTC

0 1 2 30

5

10

15

20

25

30

35

40

45

50

Days

SKLMS1 ASS1 -

ADI-PEG20 Treatment

0 1 2 30

5

10

15

20

25

30

35

400

0.01

0.05

0.1

0.5

1

Days

# o

f C

ell

(10,

000)

MNNGASS Low

0 1 2 30

5

10

15

20

25

Days

MG63ASS1 High

IC50 ug/ulCell Line ConcentrationMNNG 0.047MG63 N/ASKLMS1 0.046U2OS 0.019SKUT1 0.064SKUT1B 0.102E2 0.062E11 0.057SKES 0.056NOS N/AHuO9N2 0.056ASPS1 0.041SY0-1 0.259FUTJI 0.123HCH-1 0.042

• High ASS1 expression renders sarcoma cells resistant arginine deprivation caused by ADI-PEG20.

• Sarcoma cell lines are arginine auxotrophs

Arginine Deprivation Induces Autophagy

Autophagy. The arginine depletion using ADI-PEG20 induces autophagy by day 2 as seen by in increased LC3 cleavage and p62 alterations in ASS1 low cell lines.

MNNG/HOS ASS1 Low Xerografts

The osteosarcoma cell line MNNG/HOS was xenografted into the back fat pad of nude mice. Mice we treated daily with chloroquine and biweekly with ADI-PEG20. Tumors were measured starting on day 6. Mice were treated with PBS (Green) ADI-PEG20 (red), Chloroquine (Blue) or the combination of ADI-PEG20 and Chloroquine (Purple). The combination demonstrated statistical significance.

Enzymes

Go Both Directions

But sometimes pathways are not there

Glycolysis

TCAUrea CycleGlutamine

BiologyGlutathione

Biology

PPP

AA

Metabolism

Glycolysis

TCAGlutathione

Biology

PPP

AA

Glutamine Biology

Cancer Metabolism

Urea Cycle

UreaCycle ASS1

ADI-PEG20

AUTOPHAGY

Creation of ADI-PEG20 Resistant Cell Lines

SKLMS1 NT SKLMS1 LTAT0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

NT

ADI

P = 0.0013

P = NS

SKLMS1 WT (ASS1 Low) UntreatedSKLMS1 WT (ASS1 Low) +ADI-PEG20SKLMS1 LTAT (ASS1 High) UntreatedSKLMS1 LTAT (ASS1 High) + ADI-PEG20

Global Metabolic Approach

P = 0.0010

P = 0.0026

P = NS P = NS

P = NS

P = 0.0008

0 1 2 30

50

100

150

200

250

300

350

Days after treatment

Cel

l C

ou

nt

(*10

^4

cell

s)

SKMEL2 SKLMS1 WT SKLSM1 LTAT SKUT1

D10 -Glucose

0 1 2 30

50

100

150

200

250

300

350

Days after treatment0 1 2 3

0

50

100

150

200

250

300

350

Days after treatment0 1 2 3

0

50

100

150

200

250

300

350

D10 - glucose

D10

Days after treatment

Glucose Sensitivity

SKMEL2 SKLMS1 WT SKLMS1 LTAT SKUT10

20

40

60

80

100

120

140

NT

Oligomycin

Cell Lines Sort With Oligomycin

P = NS

P = 0.0006

P < 0.0001

P < 0.0001

Warburg Effect

• In oncology, the Warburg effect is the observation that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria as in most normal cells.

• The latter process is aerobic (uses oxygen). Malignant, rapidly growing tumor cells typically have glycolytic rates up to 200 times higher than those of their normal tissues of origin; this occurs even if oxygen is plentiful.

Glycolysis Pentose Phosphate Pathway

Purine SynthesisGlucose 6-

Phosphate dehydrogenase

6-Phosphogluconate dehydrogenase

SKLMS1 LTAT

SKLMS1 WT

SKUT1 SKMEL2

- + - + - + - +

1.535 1.789 1.291 1.182 0.869 1.045 1.913 2.571

ADI

G6PD

Actin

Intensity relative to Actin

This is not due to a Loss of G6PD

• In March 2008, Lewis C. Cantley and colleagues at the Harvard Medical School announced they had identified the enzyme that gave rise to the Warburg effect.

• PKM2, a form of the pyruvate kinase enzyme, is produced in all rapidly dividing cells, and is responsible for enabling cancer cells to consume glucose at an accelerated rate; on forcing the cells to switch to pyruvate kinase's alternative form by inhibiting the production of tumor M2-PK, their growth was curbed.

PKM2

SKLMS1 LTAT SKLMS1 WT SKUT1 SKMEL2

NT +ADI NT +ADI NT +ADI NT +ADI

PKM2

P-PKM2

PKM1

ASS1

Actin

PKM2 is a target of Autophagy

Lactate Levels

SKLMS1

High N

T

SKLMS1

High +

ADI

SKLMS1

Low N

T

SKLMS1

Low +

ADI

SKUT1 NT

SKUT1 + A

DI

SKMEL2

NT***

SKMEL2

+ A

DI***

0

0.01

0.02

0.03

0.04

0.05

0.06

Lactate Levels

H2O NH3

NH3Pi

Glutamine synthetase

Glutaminase

Glutamate dehydrogenase

H2O NH4+

TCACycle

GLUTAMINE SWITCH

0 1 2 30

50

100

150

200

250

300

350

Days after treatment

Cel

l C

ou

nt

(*10

^4

cell

s)

SKMEL2 SKLMS1 WT SKLMS1 LTAT SKUT1

D10 -Glutamine

GLUATAMINE

0 1 2 30

50

100

150

200

250

300

350

Days after treatment0 1 2 3

0

50

100

150

200

250

300

350

Days after treatment0 1 2 3

0

50

100

150

200

250

300

350

D10 - glutamine

D10

Days after treatment

GLS is Up-Regulated

sh GFP shGLS1 shGLS2 sh GFP shGLS1 shGLS2 sh GFP shGLS1 shGLS2 sh GFP shGLS1 shGLS2SKMEL2 SKLMS1 WT SKLMS1 LTAT SKUT1

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

NT

-gluc

GLS Knockdown with Glucose Withdrawal

P = 0.0012

P < 0.0001

P = 0.00295

P = 0.0404

P = 0.0027

P < 0.0001

P = NS

P = NS

GLS

Act

in

SKMEL2 SKLMS1 WT SKLMS1 LTAT SKUT10.0

0.2

0.4

0.6

0.8

1.0

1.2

NT

BPTES

ADI

ADI + BPTES

GLS INHIBITION WITH ADI

P = 0.0015

P = 0.0001

P < 0.0001

P = 0.00295

P = 0.0094

P = NS

P = 0.0030

P = 0.0007

P = 0.0008

P = 0.0259

P = 0.0013

P = 0.04

P = 0.0005

P = 0.0049

P = 0.0077

P = NS

P = NS

P = 0.043

P = NS

P = NS

In Vivo Metabolic Inhibition

0 2 4 6 8 10 12 14 16 180

20

40

60

80

100

120

140

160

180

shGFP

Poly-nomial (shGFP)

shGLS1

Glycolysis

TCAGlutathione

Biology

PPP

AA

Glutamine Biology

Cancer Metabolism

Urea Cycle

20

Glycolysis

TCAUrea CycleGlutathione

Biology

PPP

AA

Glutamine Biology

Cancer Metabolism

ADI-PEG20

ADI-PEG20

20

Glycolysis

TCAUrea CycleGlutathione

Biology

PPP

AA

Glutamine Biology

Cancer Metabolism

ADI-PEG20

ADI-PEG20BPTES

ADI-PEG 20 Induced Autophagy In ASS1 Deficient Cells

PKM2

Warburg Effect

GLS1

Glutamine

Glutamate

GSSG

GSH

Free Radical Damage Repair

TCA

ATP Generated via Oxidative Phosphorylation

Myc

ATP Generated Via Anaerobic Glycolysis

ASS1

Figure 7

Acknowledgements

• Van Tine Laboratory– Jeff Kremer– Greg Bean, Ph.D.– Matt Schulte– Sara Lange, M.D.– Philip Boone– David Chen, M.D., Ph.D.

• Cleveland Clinic– Brian P. Rubin– Munir R. Tanas

• Polaris– John Bomalaski

• Shunqiang Li• Loren Michel• Denise Reinke• Bob Maki• Sant Chawla• Robin Jones• Bill Tap