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SIMULTANEOUS AUTOPHAGY INDUCTION AND INHIBITION INDUCES CELL DEATH THROUGH NECROPTOSIS IN SARCOMAS THAT LACK ARGININOSUCCINATE SYNTHETASE 1 EXPRESSION . Disclosures. Caris Bayer DFINE. Original Observation in Osteosarcoma. Kobayashi et. al. Mol Cancer Ther 2010;9:535-544.

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Disclosures

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  1. SIMULTANEOUS AUTOPHAGY INDUCTION AND INHIBITION INDUCES CELL DEATH THROUGH NECROPTOSIS IN SARCOMAS THAT LACK ARGININOSUCCINATE SYNTHETASE 1 EXPRESSION

  2. Disclosures • Caris • Bayer • DFINE

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

  4. ArgininosuccinateSynthetase 1 • 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. HCO3+ NH4+ 2ATP Aspartate CPSI N-acetylglutamate • Citrulline Carbamyl Phosphate OTC ASS1 • Argininosuccinate • Ornithine ASL ARG Urea Fumarate • Arginine

  6. 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

  7. ASS1 Expression in Cell Lines 13/15 86.7%- Expression of ASS1 in sarcoma cell lines. 3/5 osteosarcoma cell lines lack strong expression of ASS1, with the MG63 cell line expressing the highest amount. All 3 LMS cell lines lack high ASS1 expression, as do Ewing’s,Chondrosarcoma, Sunovial and Avelolar Soft Parts Sarcoma cell lines. All expression is normalized to MG63 and then to Actin.

  8. ADI-PEG20 Treatment • High ASS1 expression renders sarcoma cells resistant arginine deprivation caused by ADI-PEG20. • Sarcoma cell lines are arginine auxotrophs

  9. Knockdown and Re-expression Experiments Demonstrate that ASS1 Is Necessary and Sufficient for Arginine Auxotrophy in Sarcoma shGFPshLuC shASS1-1 shASS1-2 Puro-MSCV MCSV-ASS1 ASS1 ACTIN ASS1 ACTIN

  10. Cell Cycle Inhibition is caused by treatment of ASS1 low cells with ADI-PEG20

  11. Autophagy • Autophagy is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. • It is a tightly regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. • It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes.

  12. 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.

  13. Duel Induction and Inhibition of Autophagy leads to cell Death. A B Induction of autophagy with ADI-PEG20. A. Cell counts over three days in cells treated with control, ADI-PEG20, chloroquine of the combination. B. Annexin V FACS on day 3 of cells treated with control, ADI-PEG20, chloroquine of the combination.

  14. Colony Formation Assays p=0.003 p=0.001 Colony formation: Colony formation assays for the ASS1 Low SKLMS1 cell line (Left) and the ASS1 High (Right) cell lines. Cells are treated with drug for 7 days and then released so that viable cells can grow out. The combination of ADI-PEG20 and Chloroquine is superior in ASS1 low cells.

  15. MNNG/HOS ASS1 Low Xenografts 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.

  16. ASS1 - SKLMS1 Xenografts PBS Chloroquine ADI-PEG20 ADI + Chloro

  17. Cell Death Pathways NT ADI CQ ADI+CQ FL RIP1 Cleaved RIP1 CASP 8 BCL2 cIAP Cleaved PARP CASP3 Cleaved CASP3 Actin

  18. Combination Treatment and Necroptosis The cell death induced by the combination of ADI-PEG20 and Chloroquine is best inhibited by necrostatin implicating necroptosis as the mechanism of cell death.

  19. FADD RIP1 RIP1 Casp-8 cIAP Ub-RIP1 Casp-3 Cleaved Casp-3 26S Proteasome • Apoptosis

  20. Necroptosis is activated by the combination of ADI-PEG20 and Chloroquine Pre-IP RIP1-IP Con ADI Chloro ADI/Chloro Con ADI Chloro ADI/Chloro RIP1 Caspase 8 RIP3 Actin RIP3 immunoprecipitates with RIP1 after treatment with chloroquine and ADI-PEG20 as part of necroptosis

  21. FADD FADD RIP3 RIP1 RIP1 Casp-8 Casp-8 cIAP • NEC-1 • Z-VAD Ub-RIP1 Casp-3 ROS Cleaved Casp-3 26S Proteasome • Apoptosis • Necroptosis

  22. Acknowledgements • Shunqiang Li • Tom Kitchens • Loren Michel • Dwight Towler • Douglas Adkins • Matthew Ellis • Van Tine Laboratory • Greg Bean, Ph.D. • Dean Weich • Philip Boone • David Chen, M.D., Ph.D. • Cleveland Clinic • Brian P. Rubin, M.D., Ph.D. • Munir R. Tanas, Ph.D. • Polaris • John Bomalaski Postdoctoral position available !!!!

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