a metabolic approach to sarcoma therapy
DESCRIPTION
A Metabolic Approach to Sarcoma Therapy . 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. - PowerPoint PPT PresentationTRANSCRIPT
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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
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Disclosures
• Caris• DFINE• Polaris• AB Science• GSK• Novartis
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Osteosarcoma
Kobayashi et. al. Mol Cancer Ther 2010;9:535-544
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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.
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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%-
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CitrullineCarbamylPhosphate
N-acetylglutamateCPSI
HCO3 + NH4 + 2ATP
Argininosuccinate
ASS
Aspartate
ArginineFumarate
ASL
Ornithine
Urea
ARG
OTC
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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
# of
Cel
l (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
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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.
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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.
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Enzymes
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Go Both Directions
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But sometimes pathways are not there
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Glycolysis
TCAUrea Cycle Glutamine Biology
Glutathione Biology
PPP
AA
Metabolism
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Glycolysis
TCAGlutathione
Biology
PPP
AA
Glutamine Biology
Cancer Metabolism
Urea Cycle
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UreaCycle ASS1
ADI-PEG20
AUTOPHAGY
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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
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SKLMS1 WT (ASS1 Low) UntreatedSKLMS1 WT (ASS1 Low) +ADI-PEG20SKLMS1 LTAT (ASS1 High) UntreatedSKLMS1 LTAT (ASS1 High) + ADI-PEG20
Global Metabolic Approach
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P = 0.0010
P = 0.0026
P = NS P = NS
P = NS
P = 0.0008
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0 1 2 30
50
100
150
200
250
300
350
Days after treatment
Cell
Coun
t (*1
0^4
cells
)
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 - glucoseD10
Days after treatment
Glucose Sensitivity
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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
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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.
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Glycolysis Pentose Phosphate Pathway
Purine SynthesisGlucose 6-
Phosphate dehydrogenase
6-Phosphogluconate dehydrogenase
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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
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• 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
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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
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Lactate Levels
SKLMS1 H
igh N
T
SKLMS1 H
igh + A
DI
SKLMS1 L
ow N
T
SKLMS1 L
ow + A
DI
SKUT1 NT
SKUT1 + A
DI
SKMEL2 N
T***
SKMEL2 + A
DI***
00.010.020.030.040.050.06
Lactate Levels
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H2O NH3
NH3Pi
Glutamine synthetase
Glutaminase
Glutamate dehydrogenase
H2O NH4+
TCACycle
GLUTAMINE SWITCH
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0 1 2 30
50
100
150
200
250
300
350
Days after treatment
Cell
Coun
t (*1
0^4
cells
)
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 - glutamineD10
Days after treatment
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GLS is Up-Regulated
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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
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SKMEL2 SKLMS1 WT SKLMS1 LTAT SKUT10.0
0.2
0.4
0.6
0.8
1.0
1.2
NTBPTESADIADI + 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
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In Vivo Metabolic Inhibition
0 2 4 6 8 10 12 14 16 180
20
40
60
80
100
120
140
160
180
shGFPPoly-nomial (shGFP)shGLS1
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Glycolysis
TCAGlutathione
Biology
PPP
AA
Glutamine Biology
Cancer Metabolism
Urea Cycle
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20
Glycolysis
TCAUrea CycleGlutathione
Biology
PPP
AA
Glutamine Biology
Cancer Metabolism
ADI-PEG20
ADI-PEG20
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20
Glycolysis
TCAUrea CycleGlutathione
Biology
PPP
AA
Glutamine Biology
Cancer Metabolism
ADI-PEG20
ADI-PEG20 BPTES
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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
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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