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A Metabolic Approach to Sarcoma Therapy

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.

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A Metabolic Approach to Sarcoma Therapy

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  1. A Metabolic Approach to Sarcoma Therapy

  2. Disclosures • Caris • DFINE • Polaris • AB Science • GSK • Novartis

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

  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. ASS1 Immunohistochemistry on 701 Soft Tissue Tumors 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 DesmoidFibromatosis 0/23 Desmoplastic Melanoma 1/10 DermatofibrosarcomaProtuberans 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/MalignantFibrousHistiocytoma 7/60 Angiosarcoma 0/5 EmbryonalRhabomdyosarcoma 1/3 Extraskeletal Myxoid Chondrosarcoma 5/7 AlveolarRhabomdyosarcoma 0/2 Nodular Fasciitis 0/6 PleomorphicRhabomdyosarcoma 2/8 SolitaryFibrous Tumor/Hemangiopericytoma 16/19 TenosynovialGiant Cell Tumor 2/34 Bone Tumors Angiomyolipoma 3/8 Giant Cell Tumor of Bone 0/7 GlomusTumor 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

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

  7. ADI-PEG20 Treatment • 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

  11. Go Both Directions

  12. But sometimes pathways are not there

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

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

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

  19. Glucose Sensitivity

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

  21. 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.

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

  23. This is not due to a Loss of G6PD ADI Intensity relative to Actin G6PD Actin

  24. 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.

  25. PKM2 is a target of Autophagy

  26. Lactate Levels

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

  28. GLUATAMINE

  29. GLS is Up-Regulated

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

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

  32. In Vivo Metabolic Inhibition

  33. Cancer Metabolism PPP Glycolysis AA Urea Cycle Glutathione Biology Glutamine Biology TCA

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

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

  36. GSSG Figure 7 Free Radical Damage Repair Glutamine GSH GLS1 Myc ASS1 Glutamate ADI-PEG 20 Induced Autophagy In ASS1 Deficient Cells TCA PKM2 ATP Generated Via Anaerobic Glycolysis ATP Generated via Oxidative Phosphorylation Warburg Effect

  37. Acknowledgements • Shunqiang Li • Loren Michel • Denise Reinke • Bob Maki • SantChawla • 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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