Presentation on theme: "SIMULTANEOUS AUTOPHAGY INDUCTION AND INHIBITION INDUCES CELL DEATH THROUGH NECROPTOSIS IN SARCOMAS THAT LACK ARGININOSUCCINATE SYNTHETASE 1 EXPRESSION."— Presentation transcript:
1SIMULTANEOUS AUTOPHAGY INDUCTION AND INHIBITION INDUCES CELL DEATH THROUGH NECROPTOSIS IN SARCOMAS THAT LACK ARGININOSUCCINATE SYNTHETASE 1 EXPRESSIONPhilip A. Boone1, Dean Weich1, Shunqiang Li1, Munir R. Tanas3, Robert Kitchens1, David Y. Chen1, Douglas R. Adkins1, John Bomalaski2, Brian P. Rubin3, Brian A. Van Tine11. 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.
3Original Observation in Osteosarcoma Kobayashi et. al. Mol Cancer Ther 2010;9:
4Argininosuccinate 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.
7ASS1 Expression in Cell Lines 13/1586.7%-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.
8ADI-PEG20 TreatmentIC50ug/ulCell LineMNNG0.047MG63N/ASKLMS10.046U2OS0.019SKUT10.064SKUT1B0.102E20.062E110.057SKES0.056NOSHuO9N2ASPS10.041SY0-10.259FUTJI0.123HCH-10.042High ASS1 expression renders sarcoma cells resistant arginine deprivation caused by ADI-PEG20.Sarcoma cell lines are arginine auxotrophs
9Knockdown and Re-expression Experiments Demonstrate that ASS1 Is Necessary and Sufficient for Arginine Auxotrophy in SarcomashGFP shLuC shASS1-1 shASS1-2Puro-MSCV MCSV-ASS1ASS1ACTINASS1ACTIN
10Cell Cycle Inhibition is caused by treatment of ASS1 low cells with ADI-PEG20
11AutophagyAutophagy 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.
12Arginine 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.
13Duel Induction and Inhibition of Autophagy leads to cell Death. A BInduction 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.
14Colony Formation Assays p= p=0.001Colony 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.
15MNNG/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.
16ASS1 - SKLMS1 XenograftsPBS ChloroquineADI-PEG20 ADI + Chloro
17Cell Death Pathways FL RIP1 Cleaved RIP1 CASP 8 BCL2 cIAP Cleaved PARP NT ADI CQ ADI+CQFL RIP1Cleaved RIP1CASP 8BCL2cIAPCleaved PARPCASP3Cleaved CASP3Actin
18Combination 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.
20Necroptosis is activated by the combination of ADI-PEG20 and Chloroquine Pre-IPRIP1-IPCon ADI Chloro ADI/Chloro Con ADI Chloro ADI/ChloroRIP1Caspase 8RIP3ActinRIP3 immunoprecipitates with RIP1 after treatment with chloroquine and ADI-PEG20 as part of necroptosis
22Acknowledgements Shunqiang Li Tom Kitchens Loren Michel Dwight Towler Douglas AdkinsMatthew EllisVan Tine LaboratoryGreg Bean, Ph.D.Dean WeichPhilip BooneDavid Chen, M.D., Ph.D.Cleveland ClinicBrian P. Rubin, M.D., Ph.D.Munir R. Tanas, Ph.D.PolarisJohn BomalaskiPostdoctoral position available !!!!