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: 314-747-8475 Pager:  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.

619/701 88.3%- ASS1 Immunohistochemistry on 701 Soft Tissue Tumors   Malignant Peripheral Nerve Sheath Tumor (NF1 Related) 3/44 Leiomyoma 0/20 Malignant Peripheral Nerve Sheath Tumor (Non-NF1) 3/31 Myxofibrosarcoma (Myxoid MFH) 0/7 Neurofibroma 0/19 Well-Differentiated Liposarcoma 0/6 Plexiform Neurofibroma 0/24 Dedifferentiated Liposarcoma 1/9 Diffuse-Type Neurofibroma 0/11 Myxoid Liposarcoma 10/12 Cellular Schwannoma 0/7 Pleomorphic Liposarcoma 1/3 Clear Cell Sarcoma 0/7 Desmoid Fibromatosis 0/23 Desmoplastic 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 0/7 Schwannoma 0/36 Epithelioid Sarcoma 2/3 Synovial Sarcoma 14/36 Low-grade Fibromyxoid Sarcoma 1/3 Gastrointestinal Stromal Tumor 3/95 Epithelioid Hemangioendothelioma 0/2 Sarcoma, NOS/Malignant Fibrous Histiocytoma 7/60 Angiosarcoma 0/5 Embryonal Rhabomdyosarcoma 1/3 Extraskeletal Myxoid Chondrosarcoma 5/7 Alveolar Rhabomdyosarcoma 0/2 Nodular Fasciitis 0/6 Pleomorphic Rhabomdyosarcoma 2/8 Solitary Fibrous Tumor/Hemangiopericytoma 16/19 Tenosynovial Giant Cell Tumor 2/34 Bone Tumors Angiomyolipoma 3/8 Giant Cell Tumor of Bone 0/7 Glomus Tumor 1/5 Fibrous Dysplasia 0/9 Granular Cell Tumor 0/4 Non-Ossifying Fibroma 1/6 Myxoma 2/5 Osteosarcoma 3/10 Leiomyosarcoma 8/56 Ewing Sarcoma/PNET 1/7 619/701 88.3%- MPNST ASS+ MPNST ASS - H&E ASS1 3+ H&E ASS1 0

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

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

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 10

Go Both Directions 11

But sometimes pathways are not there 12

Metabolism PPP Glycolysis AA Glutathione Glutamine Biology Urea Cycle TCA 13

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

AUTOPHAGY Urea Cycle ADI-PEG20 ASS1

Creation of ADI-PEG20 Resistant Cell Lines P = NS

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

P = NS P = NS P = 0.0010 P = 0.0026 P = NS P = 0.0008

Glucose Sensitivity SKMEL2 SKLMS1 WT SKLSM1 LTAT SKUT1 D10 -Glucose

Cell Lines Sort With Oligomycin P < 0.0001 P < 0.0001 P = NS P = 0.0006

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.

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

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

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.

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

Lactate Levels

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

GLUATAMINE SKMEL2 SKLMS1 WT SKLMS1 LTAT SKUT1 D10 -Glutamine

GLS is Up-Regulated

GLS Knockdown with Glucose Withdrawal P < 0.0001 P = NS P = 0.0404 P = 0.0027 P = NS P < 0.0001 P = 0.00295 P = 0.0012 GLS Actin

GLS INHIBITION WITH ADI P = 0.043 P = 0.0007 P = NS P = 0.0005 P = 0.0030 P = NS P < 0.0001 P = NS P = 0.0013 P = NS P = NS P = 0.0001 P = 0.04 P = 0.0015 P = 0.0008 P = 0.0077 P = 0.0049 P = 0.00295 P = 0.0094 P = 0.0259

In Vivo Metabolic Inhibition

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

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

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

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

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