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

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Presentation on theme: "SIMULTANEOUS AUTOPHAGY INDUCTION AND INHIBITION INDUCES CELL DEATH THROUGH NECROPTOSIS IN SARCOMAS THAT LACK ARGININOSUCCINATE SYNTHETASE 1 EXPRESSION."— Presentation transcript:

1 SIMULTANEOUS AUTOPHAGY INDUCTION AND INHIBITION INDUCES CELL DEATH THROUGH NECROPTOSIS IN SARCOMAS THAT LACK ARGININOSUCCINATE SYNTHETASE 1 EXPRESSION Philip A. Boone 1, Dean Weich 1, Shunqiang Li 1, Munir R. Tanas 3, Robert Kitchens 1, David Y. Chen 1, Douglas R. Adkins 1, John Bomalaski 2, Brian P. Rubin 3, Brian A. Van Tine 1 1. Medical Oncology, Washington University in St. Louis, St. Louis, MO, United States. 2. Polaris Group, San Diego, CA, United States. 3. Anatomic Pathology, Cleveland Clinic, Cleveland, OH, United States.

2 Disclosures Caris Bayer DFINE

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

4 Argininosuccinate Synthetase 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 Citrulline Carbamyl Phosphate N-acetylglutamateCPSI HCO 3 + NH 4 + 2ATP Argininosuccinate ASS1 Aspartate Arginine Fumarate ASL Ornithine Urea ARG OTC

6 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 Fibromatosis0/23 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 Osteosarcoma3/10 Leiomyosarcoma 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%-

7 ASS1 Expression in Cell Lines 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. 13/15 86.7%-

8 ADI-PEG20 Treatment IC50 ug/ul Cell Line MNNG0.047 MG63N/A SKLMS10.046 U2OS0.019 SKUT10.064 SKUT1B0.102 E20.062 E110.057 SKES0.056 NOSN/A HuO9N20.056 ASPS10.041 SY0-10.259 FUTJI0.123 HCH-10.042 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 ASS1 ACTIN Puro-MSCV MCSV-ASS1 ASS1 ACTIN shGFP shLuC shASS1-1 shASS1-2

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 CASP3 CASP3 Cleaved PARP 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 Casp-8 cIAP Ub-RIP1 26S Proteasome Casp-3 Apoptosis RIP1 Cleaved Casp-3 Cleaved Casp-3

20 Necroptosis is activated by the combination of ADI-PEG20 and Chloroquine Pre-IP RIP1-IP 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 Casp-8 cIAP Ub-RIP1 26S Proteasome Casp-3 Apoptosis ROS Necroptosis RIP1 FADD RIP3 RIP1 Cleaved Casp-3 Cleaved Casp-3 Casp-8 NEC-1 Z-VAD

22 Acknowledgements 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 Shunqiang Li Tom Kitchens Loren Michel Dwight Towler Douglas Adkins Matthew Ellis Postdoctoral position available !!!!


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