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A Metabolic Approach to Sarcoma Therapy

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Presentation on theme: "A Metabolic Approach to Sarcoma Therapy"— Presentation transcript:

1 A Metabolic Approach to Sarcoma Therapy
Brian A. Van Tine, M.D., Ph.D. Assistant Professor of Medicine Sarcoma Program Director SWT Tower 731 Washington University in St. Louis 660 S. Euclid Avenue Campus Box 8007 St. Louis, MO 63110 Phone:  Pager:     FAX:    

2 Disclosures Caris DFINE Polaris AB Science GSK Novartis

3 Osteosarcoma Kobayashi et. al. Mol Cancer Ther 2010;9:

4 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.

5 619/701 88.3%- ASS1 Immunohistochemistry on 701 Soft Tissue Tumors
Malignant Peripheral Nerve Sheath Tumor (NF1 Related) 3/ Leiomyoma /20 Malignant Peripheral Nerve Sheath Tumor (Non-NF1) / Myxofibrosarcoma (Myxoid MFH) /7 Neurofibroma / Well-Differentiated Liposarcoma 0/6 Plexiform Neurofibroma / Dedifferentiated Liposarcoma 1/9 Diffuse-Type Neurofibroma / Myxoid Liposarcoma /12 Cellular Schwannoma / Pleomorphic Liposarcoma 1/3 Clear Cell Sarcoma / Desmoid Fibromatosis 0/23 Desmoplastic Melanoma / Dermatofibrosarcoma Protuberans 1/6 Fibrosarcomatous Dermatofibrosarcoma Protuberans / Desmoplastic Small Round Cell Tumor 1/6 Perineurioma / Endometrial Stromal Sarcoma 0/7 Schwannoma / Epithelioid Sarcoma 2/3 Synovial Sarcoma / Low-grade Fibromyxoid Sarcoma 1/3 Gastrointestinal Stromal Tumor / Epithelioid Hemangioendothelioma 0/2 Sarcoma, NOS/Malignant Fibrous Histiocytoma / Angiosarcoma 0/5 Embryonal Rhabomdyosarcoma / Extraskeletal Myxoid Chondrosarcoma 5/7 Alveolar Rhabomdyosarcoma / Nodular Fasciitis 0/6 Pleomorphic Rhabomdyosarcoma /8 Solitary Fibrous Tumor/Hemangiopericytoma /19 Tenosynovial Giant Cell Tumor / Bone Tumors Angiomyolipoma / Giant Cell Tumor of Bone 0/7 Glomus Tumor / Fibrous Dysplasia /9 Granular Cell Tumor / Non-Ossifying Fibroma 1/6 Myxoma / Osteosarcoma 3/10 Leiomyosarcoma / Ewing Sarcoma/PNET 1/7 619/701 88.3%- MPNST ASS MPNST ASS - H&E ASS H&E ASS1 0

6 HCO3 + NH4 + 2ATP Carbamyl Phosphate N-acetylglutamate CPSI Argininosuccinate ASS Aspartate Citrulline OTC Ornithine Urea ARG Arginine Fumarate ASL

7 ADI-PEG20 Treatment IC50 ug/ul Cell Line Concentration MNNG 0.047 MG63 N/A SKLMS1 0.046 U2OS 0.019 SKUT1 0.064 SKUT1B 0.102 E2 0.062 E11 0.057 SKES 0.056 NOS HuO9N2 ASPS1 0.041 SY0-1 0.259 FUTJI 0.123 HCH-1 0.042 High ASS1 expression renders sarcoma cells resistant arginine deprivation caused by ADI-PEG20. Sarcoma cell lines are arginine auxotrophs

8 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.

9 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.

10 Enzymes 10

11 Go Both Directions 11

12 But sometimes pathways are not there
12

13 Metabolism PPP Glycolysis AA Glutathione Glutamine Biology Urea Cycle
TCA 13

14 Cancer Metabolism PPP Glycolysis AA Urea Cycle Glutathione Glutamine
Biology Glutathione Biology TCA 14

15 AUTOPHAGY Urea Cycle ADI-PEG20 ASS1

16 Creation of ADI-PEG20 Resistant Cell Lines
P = NS

17 Global Metabolic Approach
SKLMS1 WT (ASS1 Low) Untreated SKLMS1 WT (ASS1 Low) +ADI-PEG20 SKLMS1 LTAT (ASS1 High) Untreated SKLMS1 LTAT (ASS1 High) + ADI-PEG20

18

19 P = NS P = NS P = P = P = NS P =

20 Glucose Sensitivity SKMEL2 SKLMS1 WT SKLSM1 LTAT SKUT1 D10 -Glucose

21 Cell Lines Sort With Oligomycin
P < P < P = NS P =

22 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.

23 Pentose Phosphate Pathway
Purine Synthesis Glucose 6-Phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Glycolysis Pentose Phosphate Pathway

24 This is not due to a Loss of G6PD
SKLMS1 LTAT SKLMS1 WT SKUT1 SKMEL2 - + 1.535 1.789 1.291 1.182 0.869 1.045 1.913 2.571 ADI Intensity relative to Actin G6PD Actin

25 PKM2 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.

26 PKM2 is a target of Autophagy
SKLMS1 LTAT SKLMS1 WT SKUT1 SKMEL2 NT +ADI PKM2 P-PKM2 PKM1 ASS1 Actin

27 Lactate Levels

28 Glutamate dehydrogenase
GLUTAMINE SWITCH H2O NH3 H2O NH4+ TCA Cycle Glutaminase Glutamine synthetase Glutamate dehydrogenase Pi NH3

29 GLUATAMINE SKMEL2 SKLMS1 WT SKLMS1 LTAT SKUT1 D10 -Glutamine

30 GLS is Up-Regulated

31 GLS Knockdown with Glucose Withdrawal
P < P = NS P = P = P = NS P < P = P = GLS Actin

32 GLS INHIBITION WITH ADI
P = 0.043 P = P = NS P = P = P = NS P < P = NS P = P = NS P = NS P = P = 0.04 P = P = P = P = P = P = P =

33 In Vivo Metabolic Inhibition

34 Cancer Metabolism PPP Glycolysis AA Urea Cycle Glutathione Glutamine
Biology Glutathione Biology TCA 34

35 Cancer Metabolism ADI-PEG20 PPP 20 Glycolysis AA Glutathione Glutamine
Biology Glutathione Biology Urea Cycle TCA ADI-PEG20 35

36 Cancer Metabolism PPP 20 ADI-PEG20 Glycolysis AA Glutathione Glutamine
BPTES Glutamine Biology Glutathione Biology Urea Cycle TCA ADI-PEG20 36

37 ADI-PEG 20 Induced Autophagy In ASS1 Deficient Cells
Figure 7 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

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

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